During Drosophila embryogenesis, mesodermal cells are
recruited to form a stereotyped pattern of about 30
different larval muscles per hemisegment. The formation
of this pattern is initiated by the specification of a special
class of myoblasts, called founder cells, that are uniquely
able to fuse with neighbouring myoblasts. The COE transcription factor Collier plays a role in the
formation of a single muscle (muscle DA3[A] in the abdominal segments; DA4[T] in the thoracic segments T2 and T3). Col
expression is first observed in two promuscular clusters (in
segments A1-A7), corresponding to two progenitors and
then their progeny founder cells, but its transcription is
maintained in only one of these four founder cells, the
founder of muscle DA3[A]. This lineage-specific restriction
depends on the asymmetric segregation of Numb during
the progenitor cell division and involves the repression of
col transcription by Notch signaling. In col mutant
embryos, the DA3[A] founder cells form but do not
maintain col transcription and are unable to fuse with
neighbouring myoblasts, leading to a loss-of-muscle
DA3[A] phenotype. In wild-type embryos, each of the
DA3[A]-recruited myoblasts turns on col transcription,
indicating that this conversion, accomplished by the DA3[A] founder cell, induces the naive myoblasts to express founder cell distinctive patterns of
gene expression, activating col itself. Muscles DA3[A] and DO5[A] (DA4[T] and DO5[T] respectively)
derive from a common progenitor cell, the DA3[A]/DO5[A] progenitor. However, ectopic expression
of Col is not sufficient to switch the DO5[A] to a
DA3[A] fate. Together these results lead to a proposal that
specification of the DA3[A] muscle lineage requires both
Col and at least one other transcription factor, supporting
the hypothesis of a combinatorial code of muscle-specific
gene regulation controlling the formation and
diversification of individual somatic muscles (Crozatier, 1999).

Following establishment of the promuscular clusters,
specification of the progenitors is controlled by lateral
inhibition, a cell-cell interaction process mediated by the
neurogenic genes Notch (N) and Delta (Dl)). In both N and Dl mutant embryos,
promuscular Col expression is initiated normally but fails to
become restricted to a single cell per cluster, similar to
observations previously made for the expression of lsc. As a consequence, a hyperplasic expression of Col is observed from stage 11. Since it is expressed
in promuscular clusters and segregating muscle progenitors,
lsc has been proposed to play a role in muscle progenitor
selection similar to the role of achaete and scute in neuroblast
specification. However, in embryos
lacking lsc activity, selection of the Col-expressing
progenitors occurs normally at stage 11 and muscle DA3[A]
forms as in wild type (Crozatier, 1999).

The visceral muscles of the Drosophila midgut consist of syncytia and arise by fusion of founder myoblasts with fusion-competent myoblasts (fcms), as described for the somatic muscles. A single-step fusion results in the formation of
binucleate circular midgut muscles, whereas a multiple-step fusion process
produces the longitudinal muscles. A prerequisite for muscle fusion is the
establishment of myoblast diversity in the mesoderm prior to the fusion
process itself. Evidence is provided for a role of Notch signalling
during establishment of the different cell types in the visceral mesoderm,
demonstrating that the basic mechanism underlying the segregation of somatic muscle founder cells is also conserved during visceral founder cell determination. Searching for genes involved in the determination and differentiation of the different visceral cell types, two independent mutations were identified causing loss of visceral midgut muscles. In both of these mutants, visceral
muscle founder cells are missing and the visceral mesoderm consists of
fusion-competent myoblasts only. Thus, no fusion occurs resulting in a
complete disruption of visceral myogenesis. Subsequent characterization of the mutations revealed that they are novel alleles of jelly belly
(jeb) and the Drosophila Alk homolog named
milliways (miliAlk or just plain Alk). The
process of founder cell determination in the visceral mesoderm depends on Jeb signalling via the Milliways/Alk receptor. Moreover, it has been demonstrated that in the somatic mesoderm determination of the
opposite cell type, the fusion-competent myoblasts, also depends on Jeb and Alk, revealing different roles for Jeb signalling in specifying myoblast diversity. This novel mechanism uncovers a crosstalk between somatic and visceral mesoderm leading not only to the determination of different cell types but also maintains the separation of mesodermal tissues, the somatic and splanchnic mesoderm (Stute, 2004).

The process of lateral inhibition involving Notch and its ligand Delta plays a role in determining the founder myoblasts and fusion-competent myoblasts (fcms) of the somatic musculature. Since many of the processes involved in the development of the somatic musculature also seem to affect the development of the visceral muscles, whether the mechanism of determination of founder cells and fcms is also conserved was examined. In Notch mutant embryos more founder cells appear to be present in the visceral mesoderm. The visceral fcms seem to be reduced compared with the wild-type expression of sticks and stones (sns) as a marker for these cells. This reduction is
not as severe as in the somatic mesoderm but still quite obvious. In
Delta mutants, the number of founder cells also seems to be increased in comparison with the wild type and the fcms are reduced in mutant embryos (Stute, 2004).

These observations cannot exclude the possibility that the observed
phenotypes are induced by secondary effects from defects in other tissues, among others the lack of fcms in the somatic mesoderm. Therefore overexpression studies were undertaken using the UAS-GAL4 system. The GAL4 and UAS lines employed in this study also carry
rP298-lacZ, which serves to mark the founder cells. As a driver line bap-GAL4 was used to drive expression in the entire trunk visceral mesoderm. Expression of UAS-Notch+Delta, which contains the entire coding regions of both genes or UAS-Notchintra, which represents a constitutively
active form of Notch, in the visceral mesoderm, both result in a distinct phenotype. In midgut preparations of these embryos the founder cells of the circular visceral mesoderm are strongly reduced and later on, no functional visceral mesoderm can be observed. By contrast, the founder cells of the longitudinal visceral muscles, which have a different origin at the posterior tip of the
embryo, are still present. Interestingly,bap-GAL4-driven expression of the Notch ligand Delta does not result in fewer founder cells in the visceral mesoderm (Stute, 2004).

To exclude the possibility that the described defects are due to
non-endogenous effects induced by the overexpression of the examined genes in
the wrong tissue, wild-type Notch expression was analyzed and found to be expressed in the visceral mesoderm. Notch is localized at cell
membranes in the entire visceral mesoderm during stage 11, with expression becoming weaker in the fcms of the visceral mesoderm, that continue to express bap-lacZ after the determination process is finished. This reduction of Notch expression in the fcms after the establishment of the founder cells is similar to its expression in the somatic mesoderm, where Notch expression is also highest in the progenitor cell after the determination process is completed. Surprisingly, the analysis of Delta expression exhibits that this Notch ligand is not expressed in the visceral mesoderm during founder cell formation. Delta expression was found in adjacent, probably somatic cells and might be needed there to participate in the visceral determination process, as indicated by
the increased number of founder cells and reduced number of fcms in
Delta mutants. Even though Dl is expressed in the cells surrounding the visceral mesoderm, ectopic expression of UAS-Dl in these cells with a twi-GAL4 driver line does not result in an obvious phenotype, which might be due to the fact that the amount of Delta in this tissue is not the limiting factor that restricts Notch signalling. Another explanation for a missing Delta expression in the visceral
mesoderm might be that a different factor acts as a ligand for Notch in the visceral mesoderm and that the observed phenotype in Delta mutants is due to secondary effects (Stute, 2004).

Since the ectopic expression causes such a severe phenotype, the
lethality of these embryos was tested. Most of the progeny of the cross between the bap-GAL4 driver line and UAS-N+Dl or UASNintra develop and hatch but die as first larvae (78% or 70%), presumably owing to the fact that they cannot ingest any food. Ectopic expression of UAS-Dl alone also increased lethality compared with the UAS and GAL4 lines alone, but still ~65% of the larvae survive (Stute, 2004).

To confirm these results, a dominant-negative form of
Notch (UAS-dnN) was overexpressed specifically in the visceral mesoderm with a bap-GAL4 driver. The embryos exhibit an obvious duplication of most visceral founder cells but still some fcms remain (Stute, 2004).

From these results, it is concluded that Notch plays a role in the determination of the founder cells and fcms in the visceral mesoderm.
Delta, which is expressed in the cells surrounding the visceral
mesoderm, might serve as the ligand in this process but it is also possible that another factor takes over this role. Hence, not only is the fusion mechanism between the founder cells and the fcms in the somatic and visceral mesoderm conserved, but so is the initial mechanism of determination of these two cell types (Stute, 2004).

The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the mesoderm

Blood progenitors arise from a pool of pluripotential cells ('hemangioblasts') within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. This paper shows that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters ('cardiogenic clusters') that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the MAPK pathway by EGFR and FGFR is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts (Grigorian, 2011).

In this study pursued two goals: to elucidate the precise location and cellular composition of the cardiogenic mesoderm, and to analyze the mechanism by which Notch becomes activated in the restricted subset of these cells that become blood progenitors. The findings show that the cardiogenic mesoderm is comprised of segmentally reiterated pairs of clusters (cardiogenic clusters) defined by high expression levels of Dl, L'sc and activated MAPK. The MAPK pathway, activated through both EGFR and FGFR signaling, is required for the specification (EGFR) and maintenance (EGFR and FGFR) of all cardiogenic lineages. As shown previously, the default fate of all cardiogenic cells is cardioblasts. Notch activity triggered by Dl is required for the specification of blood progenitors (thoracic cardiogenic clusters) and nephrocytes (abdominal cardiogenic clusters), respectively. One of the downstream effects of MAPK signaling is to maintain high levels of Dl in the cardiogenic clusters, and to help localize Dl expression toward a dorsal subset of cells within these clusters, which will become cardioblasts. Dl stimulates Notch activity in the surrounding cells, which triggers the blood progenitor/nephrocyte fate in these cells (Grigorian, 2011).

The cardiogenic clusters form part of a larger population of mesodermal cells defined by high expression levels of l'sc. Based on l'sc in situ hybridization, these authors mapped 19 clusters of l'sc expressing cells within the somatic mesoderm. Many of these clusters (called 'myogenic clusters' in the following) give rise to one or two cells that transiently maintain high levels of l'sc, whereas the remaining cells within the cluster lose expression of l'sc. The l'sc-positive cells segregate from the mesoderm to a more superficial position, closer to the ectoderm, undergo one final mitotic division, and differentiate as muscle founder cells. Dl/Notch mediated lateral inhibition was shown to act during the singling-out of muscle founders from the myogenic clusters. Loss of this signaling pathway caused high levels of L'sc to persist in all cells of the myogenic clusters, with the result that all cells developed as muscle founders. Interestingly, loss of l'sc had only a mild effect, consisting of a slight reduction in muscle founders. This is similar to what was find in this paper in l'sc-deficient embryos, which show only a mild reduction in cardioblasts and other cardiogenic lineages (Grigorian, 2011).

The developmental fate of most of the L'sc-positive clusters within the dorsal somatic mesoderm is different from that of the ventral and lateral myogenic clusters discussed above, even though several parallels concerning the morphogenesis, proliferation, and dependence on Dl/Notch signaling are evident. The somatic (anterior, Wg-positive) mesoderm is divided into a dorsal and ventral domain based on the expression of Tin. Initially expressed at high levels in the entire mesoderm, this gene is maintained only in the dorsal mesoderm, as a result of Dpp signaling from the dorsal ectoderm. The dorsal somatic mesoderm, which is called 'early cardiogenic mesoderm', includes four L'sc-positive clusters, C2 and C14-C16. The development of C2 has been described in detail. C2 gives rise to a progenitor that divides twice; two of the progeny become the Eve-positive pericardial cells. Meanwhile, C15 which appears later at the same position as C2, seems to behave like a 'normal' myogenic cluster. It produces a progenitor that divides once and forms the founders of the dorsal muscle DA1. As shown in this paper, the two remaining dorsal clusters, C14 and C16, give rise to the cardioblasts. It is noted that the Eve-positive progenitors, as well as the cardioblasts, resemble the muscle founders derived from the typical myogenic clusters in three aspects. First, they segregate toward a superficial position, close to the ectoderm, relative to the remainder of the cells within the clusters. Secondly, they undergo one (in case of C2: two) rounds of division right after segregation. And third, they are restricted in number by Dl/Notch signaling: in all cases, they are increased in number following Dl or Notch loss of function (Grigorian, 2011).

Cardiogenic clusters, like myogenic clusters, also depend on the MAPK signaling pathway. Past studies have shown that in the Eve-positive C2 and C15 clusters, Ras, is capable of inducing the formation of additional Eve-positive progenitors. Ras is a downstream activator of both the EGFR and FGFR tyrosine kinase pathways, both of which have been seen to be important for the formation of the Eve-positive progenitors. With a loss of the FGFR pathway, Eve-positive progenitors of both the C2 and C15 cluster are lost; by contrast, the EGFR pathway affects only C15. The balanced activity of MAPK and Notch, which in part depends on reciprocal interactions between these pathways, determines the correct number of C2/C15 derived progenitors. Ras-induced MAPK activation upregulates the expression of other MAPK signaling pathway members (autoregulatory feed-back loop), but also stimulates the antagonist Argos, as well as Dl. Dl-activated Notch, in turn, inhibits MAPK signaling (Grigorian, 2011).

Both Dl/Notch and MAPK signaling are active in the C14 and C16 clusters, which constitute the definitive cardiogenic mesoderm. MAPK activity is required for the maintenance of all lineages derived from these clusters, as shown most clearly in the EGFR LOF phenotype that entails a lack of cardioblasts, blood progenitors, and pericardial nephrocytes. Overexpression of Ras results in an increased number of all three cell types, which indicates that the C14/C16 clusters attain a larger size, possibly by an additional round of mitosis. The phenotype seen in embryos suffering from loss- or overexpression of Dl/Notch pathway members can be interpreted in the framework of a classical lateral-inhibition mechanism: Dl is upregulated in the C14/C16 derived cardioblast progenitors (analogous to the Eve-progenitors of C2/C15), from where it activates Notch in the remainder of the C14/C16 cells; these cells are thereby inhibited from forming cardioblasts, and instead become nephrocytes/blood progenitors. The level of Notch activity affects the expression of tin (low Notch) and the GATA homolog srp (high Notch), which triggers the fate of cardioblasts and blood progenitors/nephrocytes, respectively (Grigorian, 2011).

MAPK is required for the initial activation of Dl in the cardiogenic clusters (just as in the myogenic clusters). Input from the pathway is most likely also instrumental in the subsequent restriction of Dl to the cardioblast progenitors. The positive interaction between MAPK and Notch signaling could occur at several levels. A mechanism shown for the ommatidial precursors of the eye disc involves Ebi and Strawberry notch (Sno), which are thought to act downstream of EGFR signaling and lead to an upregulation of Dl through the Su(H) and SMRTER complex (Grigorian, 2011).

Generally, when one progenitor cell is seen to give rise to two different cell types it is accomplished in one of two ways. One: there is an asymmetric division, where a factor expressed by the progenitor is segregated into only one daughter cell; two: a non-uniformly expressed extrinsic signal effects one cell, but not its neighboring sibling. In the posterior (abdominal) segments of the Drosophila cardiogenic mesoderm, inhibition of Notch by Numb accounts for the asymmetric activity of Notch in a small set of cardiogenic mesoderm cells, the Svp-positive cells. If Numb function is removed, these cells, which normally produce two cardioblasts and two pericardial nephrocytes, instead give rise to four cardioblasts. However, multiple nephrocytes per segment remain in numb loss-of-function mutations; furthermore, loss of numb does not cause any defect in the blood progenitors, where asymmetrically dividing Svp-positive cells are absent. This suggests that in addition to the numb-mediated mechanism, directional activation of Notch by one of its ligands is required for the majority of nephrocytes and all of the blood progenitors. It is proposed in this study that the spatially restricted upregulation/maintenance of Dl in nascent cardioblasts acts to activate Notch in the remainder of the cells within each cardiogenic cluster, which promotes their fate as blood progenitors and nephrocytes (Grigorian, 2011).

The Notch signaling pathway is typically associated with members of two different types of bHLH transcription factors. One type act as activators, while the other act as repressors. In the context of lateral inhibition, best studied in Drosophila neurogenesis, activating bHLH transcriptions factors, including genes of the AS-C like l'sc, are expressed at an early stage in clusters of ectodermal or mesodermal cells, where they activate genetic programs that promote differentiative pathways such as neurogenesis, or myogenesis/cardiogenesis. Subsequently, Notch ligands initiate the Notch pathway in these clusters; cells with high Notch activity turn on members of the Hairy/E(spl) (HES) family of bHLH genes which act as repressors and abrogate the transcriptional programs that had been set in motion by the activating bHLH factors. This paper shows that the gene cassette consisting of the Notch signaling pathway, as well as activating and repressing bHLH factors, operates in the cardiogenic mesoderm to determine the balance between cardioblasts and blood progenitors/nephrocytes. As discussed in the following, the same cassette also appears to be centrally involved in the specification of vascular endothelial cells and hematopoietic stem cells in vertebrates, which adds to the list of profound similarities between Drosophila and vertebrate blood/vascular development (Grigorian, 2011).

Even prior to the appearance of hemangioblasts, the lateral mesoderm of vertebrates is prepatterned by sequentially activated signaling pathways and transcriptional regulators similar to those that act in flies. The Wnt/Wg pathway, for example, separates subdomains of the mesoderm in vertebrates and Drosophila, as well as more ancestral ecdysozoans. Notch signaling plays an essential role in generating boundaries between segmental, as well as intra-segmental, subdomains within the ectoderm and mesoderm. The FGF signaling pathway predates the appearance of Bilaterians and plays a highly conserved role in early mesoderm patterning. Likewise, specific sets of transcriptional regulators are the targets of these signaling pathways (e.g., twist, zfh and myostatin) and play a role during the establishments of cell fate in the mesoderm. It appears, therefore, that the bilaterian ancestor featured a mesodermal subdomain, the 'cardiogenic/lateral mesoderm, in which signals of the Wg, BMP, Notch, and FGF pathways and conserved sets of transcriptional regulators established boundaries and cell fate in the mesoderm (Grigorian, 2011).

The vertebrate gene encoding an activating bHLH factor with sequence similarity to the Drosophila AS-C genes is SCL. SCL expression in the lateral mesoderm marks the first appearance of hemangioblasts; note that SCL is also expressed widely in the developing vertebrate CNS. In Zebrafish, from their site of origin in the lateral mesoderm, SCL-positive hemangioblasts migrate dorso-medially and form the intermediate cell mass (ICM). The ICM is the site of primitive endothelial blood vessel and hematopoietic cell specification. Gain of function studies carried out in zebrafish embryos have shown SCL to be one of the genes important in specifying the hemangioblast from the posterior lateral plate mesoderm. The specification of hemangioblast here comes at the expense of other mesodermal cell fates, namely the somitic paraxial mesoderm. In mice, lack of SCL affects blood and vascular development as SCL mutants are bloodless and show angiogenesis defects in the yolk sac (Grigorian, 2011).

Vertebrate homologs of the repressive Drosophila Hairy/E(spl) family of bHLH genes are the Hes and Hey (hairy/Enhancer-of-split related with YRPW motif) genes. A well studied member of the Hey family in zebrafish is gridlock, which is required for the specification of hematopoietic progenitors from the ICM. Hey 2 mutations in mice lead to severe congenital heart defects. In addition, the Hes protein plays a role in hematopoiesis as it is a positive regulator of Hematopoietic Stem Cell (HSC) expansion (Grigorian, 2011).

Genetic studies of the Notch receptors and their ligands in vertebrates support the idea that this pathway does indeed play a crucial role in the initial determination of hematopoietic stem cells. The yolk sac and the para-aortic splanchnopleura (P-Sp)/AGM (aorta gonad mesonephros) of Notch null mouse embryos lack HSCs. A similar phenotype is observed in mutants of Jagged 1, one of the Notch ligands. Notch is thought to be the deciding factor between hematopoietic and endothelial cell fates when the two originate from a common precursor or hemangioblast. In murine mutants exhibiting lower Notch1 mRNA levels, a lack of hematopoietic precursors is seen and is accompanied by an increase in the number of cells expressing endothelial cell markers. Likewise, in Drosophila, loss of Notch is associated with an increase in cardioblast number and a loss of blood precursor cells (Grigorian, 2011).

Delta function in eye morphogenesis

The R7 photoreceptor, a unique cell type within the Drosophila ommatidium, was initially proposed to be
specified by two distinct signals from neighboring cells, one from the R8 photoreceptor and another from the
R1/6 photoreceptor pair. The R8-to-R7 signal is the transmembrane ligand Bride of Sevenless (Boss), which is
received by the receptor tyrosine kinase Sevenless (Sev) and transduced via Ras activation within the
presumptive R7 cell. However, the identity of the R1/6-to-R7 signal has remained elusive. Evidence is presented that the transmembrane ligand Delta (Dl), expressed by the R1/6 pair, activates the receptor Notch
(N) in the presumptive R7 cell and constitutes the postulated R1/6-to-R7 signal required in combination with the
Boss/Sev signal to specify the R7 fate (Tomlinson, 2001).

To investigate a role for Delta/Notch signaling in R7 specification, 'experimental' clones of Dl- cells marked by the white (w) mutation were generated, as well as 'control' clones of wild-type cells similarly marked by w, and the genotypes and phenotypes of mosaic ommatidia in the adult eye were scored. Ommatidia, which are entirely Dl-, are grossly abnormal, as are the majority of mosaic ommatidia in which many of the cells are Dl-. However, the remaining mosaic ommatidia develop a normal complement of photoreceptors. 209 such normally composed ommatidia were examined to determine whether any particular photoreceptors or combinations thereof are underrepresented in Dl- mosaic tissue compared with control mosaic tissue (marked only by the w- mutation). Such underrepresentation would indicate that Delta expression is required in these cells for correct specification of the photoreceptor cell pattern. Two such examples of underrepresentation were found. In the first instance, only one of the 209 ommatidia scored contained a mutant R8 cell, and only a few contained mutant R2 (8/209) or mutant R5 (28/209) cells. The absence of mutant R8 cells probably reflects a critical role of Delta in the initial establishment of the R8 cell, and the low numbers of ommatidia with mutant R2 and R5 cells likely reflect their close lineage relationship with R8 (Tomlinson, 2001).

In the second example, and of particular relevance to this study, none of the 209 ommatidia scored were mutant for both the R1 and R6 cells, in contrast to the control experiment in which both the R1 and R6 are marked in 34% of the ommatidia. However, all other possible Dl- mosaic combinations of the R1/6/7 trio occurred at frequencies similar to those of control (wild-type) clones. It is concluded that at least one of the two members of the R1/6 pair must express Delta for the ommatidium to form a normal pattern. Because the R1/6 photoreceptors are specified after all of the other photoreceptors except the R7, this result suggests that Delta activity in these cells is specifically required for the correct specification of the R7 (Tomlinson, 2001).

In wild-type ommatidia, all of the outer photoreceptors (R1-R6) have characteristically large rhabdomeres that extend the depth of the retina, whereas R7 has a small rhabdomere restricted to the more apical regions of the retina. Among the abnormally composed ommatidia that are mosaic for Dl-, a distinct class can be identified that is identical to phenotypically normal ommatidia except that all three cells in the normal positions of R1, R6, and R7 have large rhabdomeres that extend the depth of the retina. The individual identities of the cells in these ommatidia were inferred from the relative positions of the photoreceptors compared with the surrounding ommatidia; the position of the R8 cell (normally situated basally and between R1 and R2) was used to corroborate the accuracy of this assessment. In such ommatidia, both R1 and R6 positions were invariably found to be Dl-. Thus, it appears that the absence of Delta function in both R1 and R6 prevents the normal specification of R7 and that the cell in the R7 position develops instead as an outer photoreceptor. The R8 cell was wild-type in all 30 of these ommatidia, confirming the finding that the requirement for Delta signaling for the correct specification of R7 cannot be satisfied by Delta derived from R8 (Tomlinson, 2001).

Delta therefore has the behavior expected for an R1/6-derived signal that is required to specify the R7 fate. Moreover, its role is distinct from that of the R8-to-R7 signal because in the absence of Delta signaling, the presumptive R7 cell chooses an outer photoreceptor fate rather than the cone cell fate chosen when Boss/Sev signaling is compromised. It is likely that the particular outer photoreceptor fate chosen is that of R1/6 (Tomlinson, 2001).

Specification of the Drosophila R7 photoreceptor has emerged as a central paradigm for how a single cell chooses its fate based on signals received from neighboring cells. However, even in this apparently simple case, considerable uncertainty persists about the number of signals involved as well as the roles of these signals. Two signals were initially proposed, based in large part on the observation that the presumptive R7 cell makes a unique set of contacts with the R8 cell and both members of the R1/6 pair. However, only one signal, the Boss/Sev signal sent from R8 to R7, has been identified, and in the absence of any evidence for a second signal, the consensus view in the field has shifted toward one-signal rather than two-signal models. The finding that Notch activation is a necessary signal for R7 specification does not concur with reports that ectopic activation of the Notch pathway in the presumptive R7 cell causes it to develop inappropriately as a cone cell. However, these reports describe experiments using entirely mutant sev-Nintra ommatidia in which the Notch pathway is constitutively activated in several cells within each ommatidium. Under these conditions, many cells choose inappropriate fates, and the ommatidia undergo aberrant rotations and exhibit other disorders, precluding an accurate assessment of the fate of the presumptive R7 cell (Tomlinson, 2001).

One-signal models for R7 specification have been supported in large part by experiments that show that the forced activation of the Ras pathway in the presumptive cone cells appears sufficient to direct them to the R7 fate. However, Delta is expressed in the presumptive R3/4, R1/6, and R7 photoreceptors, which directly contact the presumptive cone cells. Hence, the presumptive cone cells normally receive a Notch input. Accordingly, forced activation of the Ras pathway in these cells would provide the second of the two inputs predicted by the two-signal model to specify the R7 fate. Thus, the experimental finding that forced Ras activation causes presumptive cone cells to choose the R7 fate is compatible with the two-signal model (Tomlinson, 2001).

Another challenge to the two-signal model is the evidence that Ras activity is necessary for the normal specification of cone cells. It has been reported that two genes, sparkling and prospero, show differential expression in the cone cells relative to the R7 cells and are subject to direct regulation by the Ras pathway in cone cells. Taken together with previous observations suggesting that cone cell specification depends on activation of the Drosophila EGF receptor, these results present a paradox. If the choice of the cone cells fate normally requires Ras activation, why does forced activation of the Ras pathway in these cells cause them to develop as ectopic R7s? It is suggested that the R7 and cone cell precursor choose distinct cell fates because they receive different levels of Ras input, higher for the R7 fate (via Boss/Sev signaling) and lower for the cone cell fate (Tomlinson, 2001).

Following the establishment of the ommatidial precluster composed of the R8 cell and adjacent R2, R3, R4, and R5 cells, the surrounding undifferentiated cells undergo a wave of division, giving rise to a distinct population from which the R1/6, R7, and cone cells will be recruited. Unlike the precursor cells that give rise to the precluster, all of the cells within this second population express the transcription factor Lozenge, which appears to distinguish their response to Notch and Ras activation from that of the first round of precursor cells. Two mechanisms are suggested by which Notch and Ras signaling may function combinatorially to direct cells within this Lozenge-expressing precursor population to choose the R1/6, R7, and cone cell fates. In the first model, Notch and Ras activation are viewed as distinct and independent inputs and a simple combinatorial code is proposed for determining the fates of the Lozenge-expressing cells, which are recruited to the ommatidium after the five-cell precluster is established: (1) if a cell receives Ras activation alone, it becomes an R1/6 type; (2) if a cell receives both Ras and Notch activation, it is directed to the R7 pathway; (3) if a cell receives only Notch activation, it becomes a cone cell. It is suggested that during normal development, only the R7 precursor receives both Ras and Notch activation (from the R8 and R1/6 cell, respectively), whereas the R1/6 precursors receive only the Ras input (probably via Spitz/EGF receptor signaling from the neighboring R2/5 cells), and the cone cell precursors receive only the Notch input (presumably via Delta/Notch signaling from neighboring photoreceptors). This combinatorial code is overly simplistic, as there is evidence that cone cell specification depends on Ras activation. Accordingly, the R7 and cone cell precursors are viewed as equivalent in terms of Notch activation, but distinct in terms of Ras activation, with only the presumptive R7 receiving an extra boost in Ras activity via Boss/Sev signaling. A more accurate description of the combinatorial code might therefore be as follows: (1) high Ras alone specifies the R1/6 fate; (2) high Ras plus Notch specifies the R7 fate, and (3) moderate Ras plus Notch specifies the cone cell fate. This model accounts for all of the changes in cell fates observed when Notch or Ras inputs are abolished or ectopically provided to the R1/6, R7, or cone cell precursors (Tomlinson, 2001).

In the second model, the possibility is considered that the Notch and Ras signals are not independent but, rather, that the action of one may facilitate the action of the other. For example, Sev is normally expressed at a low level in the R1/6 precursors but at a high level in the R7 precursor. Hence, one function of Delta/Notch signaling may be to upregulate Sev expression in the R7 precursor, predisposing this cell to receive a high level of Ras activation via its contact with the Boss-expressing R8 cell. When Notch is ectopically activated in the R1/6 precursors, these cells would be induced to express high levels of Sev, and because they also contact the R8 cell, they too would receive high levels of Ras activation. In an extreme version of this model, the R1/6 and R7 fates would be distinguished solely by different levels of Ras activation, and the only role of the Notch/Delta signal from R1/6 would be to boost the level of Ras activation in the R7 precursor cell to reach the threshold necessary to specify the R7 fate. However, less extreme versions of this model are equally tenable. For example, Delta-Notch signaling might serve both to provide a distinct Notch input to the R7 precursor as well as to predispose it to receive a higher level of Ras activation than the R1/6 precursors. In this scenario, the cell-type code would be that (1) moderate Ras alone specifies the R1/6 fate, (2) high Ras with Notch specifies the R7 fate, and (3) moderate Ras with Notch specifies the cone cell fate (Tomlinson, 2001).

The Notch pathway is known to act during initiation and
differentiation of wing veins to refine the adult vein pattern. Since nemo mutant, nmoadk, was identified as a modifier of Notch in the eye, the link between nmo and Notch signaling in the wing was investigated. Genetic interactions between nmoadk
and mutations in several components of the Notch pathway were characterized. Mutations in the ligand Delta (Dl/+) cause a mild vein thickening phenotype. This phenotype is synergistically enhanced by homozygosity for nmoadk. Conversely, mutations in the
negative regulator Hairless (H/+), which normally exhibit
shortening of LV, suppress the ectopic veins seen in
nmoadk. In addition to interactions in wing
veins, H and nmoadk show a synergistic interaction in the macrochaete bristles of the head and notum (Verheyen, 2001).

nmoadk flies have a mild bristle loss phenotype, and occasionally display bent bristles or duplicated bristles. H/+
flies display a characteristic dominant loss of macrochaetes. Homozgosity for
nmoadk in a H/+ background leads to a dramatic enhancement of the H/+ bristle loss phenotype. Since nmoadk
mutations are enhanced by Dl, and are
suppressed in the wing by H, whether
nmo acts upstream of Notch was examined. It was asked if the nmoadk extra vein defect could be rescued through ectopic activation of Notch signaling. Delta and E(spl)mß were ectopically expressed with the 32B-Gal4 driver, which is expressed in the wing
blade. E(spl)mß is normally expressed in the cells flanking
the presumptive veins and acts to suppress rhomboid
expression to the narrow band of vein progenitors. Ectopic expression of UAS-E(spl)mß leads to mild vein thinning and a shortening of LV. Both UAS-Delta and UAS-E(spl)mß specifically
suppress the extra veins associated with nmoadk
mutations. Thus, both ectopic activation of
the Notch pathway and loss of a negative regulator as seen
with H1/+ can lead to suppression of ectopic veins caused
by nmoadk. These results suggest that Nemo is upstream of
Notch and acts in a common vein regulatory pathway (Verheyen, 2001).

The Drosophila rotund (rn) gene is required in the wings, antenna, haltere, proboscis and legs. Previously identified in the rotund region was a member of the Rac family of GTPases, denoted the RacGAP84C or rotund racGAP gene. However, rotund racGAP is not responsible for the rotund phenotypes. The rotund gene has now been isolated. It is a member of the Krüppel family of zinc finger genes. The adjacent roughened eye locus specifically affects the eye and is genetically separable from rotund. However, roughened eye and rotund are tightly linked, and thanks to this connection, the roughened eye transcript was isolated. Intriguingly, roughened eye is part of the rotund gene but is represented by a different transcript. The rotund and roughened eye transcripts result from the utilization of two different promoters that direct expression in non-overlapping domains in the larval imaginal discs (St Pierre, 2002 and references therein).

Little is known about the genetic cascades within which roe and rn are acting. The results from eye-antennal imaginal discs indicate that roe acts at the morphogenetic furrow, as evident both from its expression and from the effects on Delta and Scabrous expression in roe mutants. Both Dl and sca play roles in spacing the array of ommatidial preclusters in the morphogenetic furrow, and it is interesting to note that the expression of roe at the furrow is not evenly distributed and appears stronger in clusters of cells. Genetic screens for modifiers of the Nspl mutation have identified roe as an enhancer, and sca and Dl as suppressors of the Nspl eye phenotype. Given the dynamics of N signaling, these results support models where Roe acts to either positively or negatively regulate Dl and Sca. A genetic interaction screen for enhancers of glass also identified roe, an interesting finding given that ectopic expression of roe using GMR-GAL4 leads to a glass-like phenotype with a loss of bristles and pigment cells (St Pierre, 2002 and references therein).

Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it
in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a
nuclear protein (Wee1) and a cytoplasmic protein (Myt1). The genes encoding Drosophila Wee1 and Myt1 have been isolated and genetic approaches are being used to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during
eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known
regulators of the G2/M transition, suggesting that these transgenic strains can be used in modifier screens to identify potential regulators of Wee1 and Myt1. To
confirm this idea, a collection of deletions for loci that can modify the eye overexpression phenotypes was tested and several loci were identified as dominant modifiers.
Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting
that Myt1 is potentially a downstream target for Notch activity during eye development. One of the loci identified as a specific enhancer of the GMR-Dmyt1 eye phenotype is Delta. This interaction could reflect defects in Dl-dependent neuronal specification that are enhanced by GMR-Dmyt1 activity, or it may indicate a novel role for Delta/Notch signaling in regulating Myt1 activity (Price, 2002).

The roles of cis-inactivation by Notch ligands and of neuralized during eye
and bristle patterning in Drosophila

The receptor protein Notch and its ligand Delta are expressed throughout
proneural regions yet non-neural precursor cells are defined by Notch activity
and neural precursor cells by Notch inactivity. Not even Delta overexpression
activates Notch in neural precursor cells. It is possible that future neural
cells are protected by cis-inactivation, in which ligands block activation of
Notch within the same cell. The Delta-ubiquitin ligase Neuralized has been
proposed to antagonize cis-inactivation, favoring Notch activation.
Cis-inactivation and the role of Neuralized has not yet been studied in tissues
where neural precursor cells are resistant to nearby Delta, however, such as the
R8 cells of the eye or the bristle precursor cells of the epidermis.
Overexpressed ligands block Notch signal transduction cell-autonomously in
non-neural cells of the epidermis and retina, but do not activate Notch
nonautonomously in neural cells. High ligand expression levels are required for
cis-inactivation, and Serrate is more effective than Delta, although Delta is
the ligand normally regulating neural specification. Differences between Serrate
and Delta depend on the extracellular domains of the respective proteins.
Neuralized acts cell nonautonomously in signal-sending cells during
eye development, inconsistent with the view that Neuralized antagonizes
cis-inactivation in non-neural cells. It is concluded that Delta and Neuralized
contribute cell nonautonomously to Notch signaling in neurogenesis, and the
model that Neuralized antagonizes cis-inactivation to permit Notch activity and
specification of non-neural cells is refuted. The molecular mechanism rendering
Notch insensitive to paracrine activation in neural precursor cells remains
uncertain (Li, 2004).

One difference between neural and non-neural cells may be neur, which
has been proposed to relieve cis-inactivation cell autonomously by endocytosing
Dl, or to promote paracrine signaling in experiments where
neur appears nonautonomous. Neur might
make non-neural cells less sensitive to cis-inactivation, so that only high
Delta levels would be effective (Li, 2004).

Cell autonomy of neur function in the eye was investigated
using FLP-mediated mitotic
recombination in neur heterozygous larve to induce cell clones homozygous
for neur1, a loss of function neur allele. Mitotic
recombination was induced late in the third larval instar to generate small
neur mutant clones. Mosaic adult eyes were sectioned and the cellular
contribution of neur mutant cells recorded. In many cases presence of
neur mutant cells was associated with changes in the number of
photoreceptor cells. Ommatidia with too many or too few photoreceptor cells were
both observed, as for other neurogenic mutations. Less often,
ommatidia containing one or more neur mutant cells differentiated 8
photoreceptor cells in the normal arrangement. Forty such mosaic ommatidia were
examined in more detail to identify any cells where neur function might
be dispensable (Li, 2004).

Ommatidia almost never developed
normally with neur mutant R8 cells. Only a single example was found.
If neur activates N signaling by
antagonizing cis-inactivation, then one would expect that neur would be
required in cells where N is active, but dispensable where N is inactive. On
this basis neur should not be required in R8 cells. By contrast the data
suggested that R8 is where neur is most important. It has been found
that Dl is also required in R8 cells. The possibility is excluded
that either neur or Dl
is required directly in the execution of the R8 differentiation pathway
because many ectopic R8 cells differentiate in large neur or Dl
mutant clones, or when the whole eye is mutant.
Instead the data suggest that ommatidia with neur
or Dl mutant R8 cells could not develop normally because neur
acts in R8 to promote Dl-mediated activation of N in neighboring cells (Li, 2004).

To explore further
when neur acts autonomously or nonautonomously, other aspects of retinal
N signaling were also examined. During ommatidial development, Notch signaling
breaks the symmetry of the R3/R4 pair. Dl from R3 activates N in R4.
neur mutant cells were five times as likely to
take R4 fate as R3 fate. Thus neur is important for the nonautonomous
signaling activity of Dl from the R3 cell but not required autonomously for
activity of N in the R4 cell. Only rarely can a neur mutant R3 cell
activate N in a neighboring R4 cell, but neur R4 cells can be activated
in response to wild type R3 cells (Li, 2004).

Further data from abnormally
constructed ommatidia
support the importance of neur in R3. These were ommatidia where the
R3/R4 pair remained symmetrical. 17 symmetrical ommatidia were found with two R3
cells in place of R3/R4. In 3 such ommatidia both R3-like cells
were mutant for neur. In 13 of the other 14 cases the cell in
the location that should normally have become R3 was neur mutant; in a
single case the cell positioned to become R4 was neur mutant. These
symmetrical ommatidia indicate that when R3 cells lack neur function, the
neighboring cell receives insufficient Dl signaling to take R4 fate and instead
is transformed into a second R3 (Li, 2004).

N signaling is further required for
R7 specification. N is activated in R7 precursors by Dl from neighboring R1 and
R6 cells. R1 and R6 act redundantly but if both R1 and R6 are Dl
mutant then the R7 precursor adopts R1/6-like morphology. R7 was frequently
neur mutant in normally-constructed ommatidia, so neur is not
essential in the R7 precursor cell. R1 and R6 were never both mutant for
neur in normal ommatidia. One ommatidium was found in which both R1 and
R6 were neur mutant. In this ommatidium the cell in the R7 position was
wild type for neur but had R1/6-like morphology. These results indicate that
neur, like Dl, is not required for N activity in the R7 cell
itself. neur may be required nonautonomously in R1 and R6 for proper R7
specification (Li, 2004).

A screen for modifiers of notch signaling uncovers Amun, a protein with a critical role in sensory organ development

Notch signaling is an evolutionarily conserved pathway essential for many cell fate specification events during metazoan development. A large-scale transposon-based screen was conducted in the developing Drosophila eye to identify genes involved in Notch signaling. 10,447 transposon lines from the Exelixis collection were screened for modifiers of cell fate alterations caused by overexpression of the Notch ligand Delta, and 170 distinct modifier lines were identified that may affect up to 274 genes. These include genes known to function in Notch signaling, as well as a large group of characterized and uncharacterized genes that have not been implicated in Notch pathway function. A gene was further analyzed that has been named Amun, and it encodes a protein that localizes to the nucleus and contains a putative DNA glycosylase domain. Genetic and molecular analyses of Amun show that altered levels of Amun function interfere with cell fate specification during eye and sensory organ development. Overexpression of Amun decreases expression of the proneural transcription factor Achaete, and sensory organ loss caused by Amun overexpression can be rescued by coexpression of Achaete. Taken together, these data suggest that Amun acts as a transcriptional regulator that can affect cell fate specification by controlling Achaete levels (Shalaby, 2009).

Drosophila continues to play a leading role in the discovery of genes and mechanisms implicated in developmental processes mediated by, or associated with, the Notch signaling pathway. This study presents the results of a transposon screen for the effects of loss-of-function and gain-of-function mutations in a genetic background sensitized for Delta-mediated cell fate changes. In addition, Amun, a nuclear protein identified as a suppressor in the screen was characterized. Amun suppresses a dominant-negative effect of Delta overexpression on cone cell induction in the eye, suggesting that Amun can positively regulate Notch signaling in this context. Alternatively, Amun may function in a parallel or intersecting pathway to affect cone cell development. Evidence is provided that Amun can function early during the cellular patterning underlying mechanosensory bristle development by downregulating the expression of the proneural transcription factor Achaete. The identification and initial characterization of Amun function reflect the potential of the ensemble of 170 transposon insertions identified in the screen for discovery of additional factors that affect Notch signaling mediated development (Shalaby, 2009).

The Exelixis collection covers ~50% of Drosophila genes and contains many new alleles for genes that may prove to be involved in the Delta–Notch signaling pathway or other developmental pathways. The collection has also been screened in a search for modifiers of a Notch loss-of-function signaling phenotype in the wing margin using C96-driven MamDN. Among the 170 modifiers that were identified, 29 lines were also recovered by Kankel (2007) and 141 lines were recovered only in the current screen. Among the putative genes recovered in both screens are several known Notch pathway members and genes that have been previously recovered from Notch-based screens (e.g., numb, wingless, puckered, and Ras85D). In addition, several genes that had not been implicated previously in Notch signaling were identified in both screens, supporting roles for their encoded products during Notch-mediated development. These genes include peanut (a septin), Oatp30B (an ion channel), Indy (a transporter), and Hr38 (a hormone receptor). Of potentially equal interest are the 11 transposon lines that modified phenotypes in secondary tests in this work. Genes potentially disrupted by these transposons include karst (βHeavy-spectrin), bifocal (a cytoskeletal regulator), diaphanous (an actin-binding protein), and caudal (a transcriptional regulator). Further characterization of these genes, as well as other genes recovered in the screen, will help provide a deeper understanding of the mechanisms that govern the Notch signaling pathway (Shalaby, 2009).

A number of the results suggest that Amun is required for cell fate determination during Notch-mediated bristle organ development. Reduction of Amun function and Amun protein overexpression in the developing notum, using several Gal4 drivers including pnr, ptc, sca, and sr, generate defects during microchaeta and macrochaeta development. Substantial loss of microchaetae is observed in the nota of adults that express Amun under pnr-Gal4 or sr-Gal4 control during development. Immunohistochemical analysis of developing nota and the Achaete expression rescue experiments demonstrates that this loss of microchaetae is due to loss of the bHLH transcription factor Achaete. The expression patterns of the proneural proteins Achaete and Scute are best characterized for the dorsocentral macrochaetae, for which cis-regulatory elements control the expression of these genes in specific patterns to establish proneural clusters. These enhancer elements are thought to be activated directly by members of several signaling pathways, including Decapentaplegic and Wingless, as well as by other factors including Pannier (Pnr), Daughterless (Da), Chip, and members of the Iroquois complex (Araucan and Caupolican). The expression of achaete/scute is antagonized by several factors, including U-shaped and dCtBP, both of which bind Pnr to form a transcriptional corepressor complex; Extramacrochaetae (Emc), which forms a heterodimer with Da to inactivate it; and the E(spl)-C proteins, which are downstream targets of Notch signaling. In microchaeta proneural groups, Achaete is also known to be repressed by Hairy, as well as by Notch signaling. This study demonstrates that the effect of Amun overexpression on Achaete levels is cell autonomous, suggesting that the action of Amun on achaete expression could be direct. However, while it is tempting to speculate that Amun regulates Achaete levels by directly binding to cis-regulatory elements that affect achaete expression, it cannot be ruled out that Amun functions by repressing an activator of achaete (e.g., Da or Chip), by activating a repressor of achaete (e.g., Emc, Hairy, or the Notch pathway), or by destabilizing achaete mRNA or protein (Shalaby, 2009).

Reductions in Amun function by RNA interference result in small and disorganized microchaetae. In contrast to the Amun overexpression phenotype, the small microchaeta phenotype is not easily attributable to changes in Achaete expression, given that Achaete has no known roles in bristle development subsequent to SOP specification. It has been shown that bristle shaft size can be correlated with several processes. First, both the shaft and socket cells undergo endoreplication to form polyploid nuclei that are required to form the elongated shaft structure. The degree of endoreplication has been correlated with shaft size. Second, shaft length can be affected by mutations in genes that affect actin bundle formation necessary for proper elongation of the shaft. Third, there is a period of rapid protein synthesis during sensory bristle development that enables the shaft and socket cells to generate the high levels of protein required for the development of the socket and shaft structures. Genes necessary for this process include small bristles [which exports mRNA from the nucleus into the cytoplasm and the Minute loci (genes encoding ribosomal proteins), which can affect bristle shaft length. Preliminary data suggest that Amun is unlikely to affect endoreplication. Nuclei of microchaetae that develop in regions of the notum expressing sr-driven AmunRNAi were investigated and no consistent effects on nuclear size were found as compared to the nuclei of cells of microchaetae in regions devoid of AmunRNAi. Therefore the notion is favored that Amun may be required for transcriptional regulation of specific genes involved in growth and elongation of the shaft or for the elevated levels of mRNA and protein synthesis required for shaft development (Shalaby, 2009).

The finding that Amun can affect Achaete expression levels, together with the identification of Amun as a nuclear protein with a putative DNA glycosylase domain, are consistent with the hypothesis that Amun functions as a transcriptional regulator. While DNA glycosylases are best known for repair of damaged and mismatched bases, recent work indicates that they also play roles in transcriptional regulation. The mammalian DNA glycosylase thymine DNA glycosylase (TDG) acts as a transcriptional co-activator, when bound to CREB-binding protein (CBP) and p300 (Tini, 2002), to enhance CBP-activated transcription in cell culture (Cortazar, 2007). It also acts as a transcriptional corepressor when bound to thyroid transcription factor-1 (TTF1) to repress TTF1-activated transcription in cell culture (Cortazar, 2007; Kovtun, 2007). The Arabidopsis DNA glycosylase DEMETER is required to activate expression of the maternal MEDEA allele, an imprinted maternal gene essential for viability. In light of these studies, the nuclear localization of Amun is suggestive of a function for Amun as a transcriptional regulator (Shalaby, 2009).

In summary, this study demonstrated that Amun is a nuclear protein essential for organismal viability and proper cell fate specification during metamorphosis of Drosophila tissues, including the eye and mechanosensory organs. It is suggested that Amun affects at least two distinct processes during bristle organ development because of the distinct loss-of-function and gain-of-function bristle phenotypes associated with Amun. One pathway is critical for regulation of Achaete protein levels, and the other pathway affects sensory organ bristle shaft size. Because the sequence of Amun contains a putative DNA glycosylase domain, it was reasoned that Amun may act as a transcriptional regulator, as previously demonstrated for other DNA glycosylases. Further characterization of Amun is necessary to identify distinct transcriptional targets and pathways on which it may act and to decipher its potential function as a DNA glycosylase during Drosophila development (Shalaby, 2009).

Cancer is both a genetic and an epigenetic disease. Inactivation of tumour-suppressor genes by epigenetic changes is frequently observed in human cancers, particularly as a result of the modifications of histones and DNA methylation. It is therefore important to understand how these damaging changes might come about. By studying tumorigenesis in the Drosophila eye, two Polycomb group epigenetic silencers, Pipsqueak and Lola, have been identified that participate in this process. When coupled with overexpression of Delta, deregulation of the expression of Pipsqueak and Lola induces the formation of metastatic tumours. This phenotype depends on the histone-modifying enzymes Rpd3 (a histone deacetylase), Su(var)3-9 and E(z), as well as on the chromodomain protein Polycomb. Expression of the gene Retinoblastoma-family protein (Rbf ) is downregulated in these tumours and, indeed, this downregulation is associated with DNA hypermethylation. Together, these results establish a mechanism that links the Notch-Delta pathway, epigenetic silencing pathways and cell-cycle control in the process of tumorigenesis (Ferres-Marco, 2006).

Correct organ formation depends on the balanced activation of conserved developmental signalling pathways (such as the Wnt, Hedgehog and Notch pathways). If insufficient signals are received, organ growth may be deficient. By contrast, excess signalling leads to an overproduction of progenitor cells and a propensity to develop tumours. When such hyperproliferation is associated with the capacity of cells to invade surrounding tissue and metastasis to distant organs, cancer develops. Indeed, activation of the Wnt, Hedgehog and Notch pathways is a common clinical occurrence in cancers. Curiously, activation of any of these pathways in animal models seems to be insufficient for cancer to develop, indicating that synergism with other genes is required for these pathways to produce cancer (Ferres-Marco, 2006).

Cellular memory or the epigenetic inheritance of transcription patterns has also been implicated in the control of cell proliferation during development, as well as in stem-cell renewal and cancer. Proteins of the Polycomb group (PcG) are part of the memory machinery and maintain transcriptional repression patterns. The upregulation of several PcG proteins has been associated with invasive cancers. Thus, increased amounts of EZH2, the human homologue of the Drosophila histone methyltransferase E(z), is associated with poorer prognoses of breast and prostate cancers (Ferres-Marco, 2006).

Another histone methyltransferase implicated both in gene silencing and in cancer is SUV39H1, a homologue of Drosophila Su(var)3-9. SUV39H1 and Su(var)3-9 methylate histone H3 on lysine 9 (H3K9me), and this epigenetic tag is characteristic of heterochromatin and DNA sequences that are constitutively methylated in normal cells. DNA methylation is another mechanism involved in cellular memory that actively contributes to cancer. Indeed, numerous tumour-suppressor genes, including the retinoblastoma gene RB, are silenced in cancer cells by DNA hypermethylation. Inactivation of the RB tumour-suppressor pathway is considered an important step towards malignancy; thus, it is important to understand how these damaging epigenetic changes are initiated in cells that become precursors of cancer. Moreover, it is equally important to determine the connection between these processes and the developmental pathways controlling proliferation (Ferres-Marco, 2006).

Forward genetic screening in suitable animals is a powerful tool with which to identify tumour-inducing genes and to reveal changes that precede neoplastic events in vivo. The developing eye of Drosophila melanogaster is a good model for such studies because it is a simple and genetically well-defined organ. The growth of the eye depends on Notch activation in the dorsal-ventral organizer by its ligands Delta (human counterparts, DLL-1, -3, -4) and Serrate (human counterparts, JAGGED-1, -2). This study used the 'large eye' phenotype, produced by overexpression of Delta, as a tool to screen for mutations that interact with the Notch pathway and convert tissue overgrowths into tumours. One mutation, eyeful, was isolated that combined with Delta induces metastatic tumours. eyeful forces the transcription of two hitherto unsuspected growth and epigenetic genes, lola and pipsqueak (psq). The identification of eyeful has been a starting point from which to unravel crosstalk between the Notch and epigenetic pathways in growth control and tumorigenesis. The fact that many epigenetic factors are involved in cancer suggests that these processes may be more generally involved in tumorigenesis than at first it might seem (Ferres-Marco, 2006).

To identify genes that interact with the Notch pathway and that influence growth and tumorigenesis, the Gene Search (GS) system was used to screen for genes that provoked tumours when coexpressed with Delta in the proliferating Drosophila eye. The ey-Gal4 line was used for both eye-specific and ubiquitous induction, resulting in the transactivation of UAS-linked genes throughout the proliferating eye discs. It was through such a screen that the GS88A8 line was isolated. Generalized overexpression of Delta by ey-Gal4 (hereafter termed ey-Gal4 > Dl) produces mild eye overgrowth. In most of the flies in which the GS88A8 line was coexpressed with Delta, tumours developed in the eyes. Moreover, in ~30% of the mutant flies, secondary eye growths were observed throughout the body, and in some flies the whole body filled up with eye tissue. These secondary eye growths had ragged borders, indicating invasion of the mutant tissue into the surrounding normal tissue. As a result, the GS88A8 line was named 'eyeful' (Ferres-Marco, 2006).

A developmental analysis of the tumours was undertaken. To facilitate analyses, a triple mutant strain was generated carrying the eyeful, UAS-Dl and ey-Gal4 transgenes all on the same chromosome (ey-Gal4 > eyeful > Dl. In this strain, mutant eye discs showed massive uncontrolled overgrowth (some discs were more than five times their normal size). In most discs, the epithelial cells had lost their apical-basal polarity, and some had a disrupted basement membrane and grew without differentiating (Ferres-Marco, 2006).

These results were extended to the wing disc. (1) dpp-Gal4 was used to direct coexpression of eyeful and Delta along the anterior-posterior boundary of the wing (perpendicular to the endogenous Delta domain along the dorsal-ventral boundary. In a normal wing disc, the dpp-Gal4 driver typically establishes a stripe of green fluorescent protein (GFP) expression with a sharp border at the boundary. Whereas wild-type (or single eyeful) cells expressing GFP conformed with this pattern, some of the eyeful and Delta cells were found outside this stripe, indicating that the mutant cells can disseminate and invade adjacent regions of the disc. (2) The MS1096-Gal4 line was used to direct expression in the dorsal wing disc compartment. Under these conditions, the wing tissue grew massively and aggressively, and the mutant tissue failed to differentiate. These observations suggest that, when coupled with Delta overexpression, an excess of the gene products flanking the eyeful insertion site induces the formation of tumours capable of metastasising (Ferres-Marco, 2006).

The genomic DNA flanking the eyeful P-element was isolated and sequenced. eyeful is inserted in an intron of the gene longitudinals lacking (lola), which is known to be a chief regulator of axon guidance. lola encodes 25 messenger RNAs that are produced by alternative splicing and that generate 19 different transcription factors. All of the different isoforms share four exons that encode a common amino terminus, which contains a BTB or POZ domain. In addition, all but one of these transcription factors are spliced to unique exons encoding one or a pair of zinc-finger motifs (Ferres-Marco, 2006).

The GS P-elements allow Gal4-dependent inducible expression of sequences flanking the insertion site in both directions. The nearest gene in the opposite direction to transcription of lola is the psq gene. This gene encodes nine variants produced by alternative splicing and alternative promoter use, generating four different proteins. Three of the psq isoforms contain a BTB or POZ domain in the N terminus, and a histidine- and glutamine-rich region downstream of this domain. Two of the BTB-containing isoforms and the isoform that lacks this domain contain four tandem copies of an evolutionarily conserved DNA-binding motif, the Psq helix-turn-helix (HTH) motif (Ferres-Marco, 2006).

psq was initially identified for its 'grandchildless' and posterior group defects and was subsequently shown to have a role in retinal cell fate determination. Psq is essential for sequence-specific targeting of a PcG complex that contains histone deacetylase (HDAC) activity. Psq binds to the GAGA sequence, which is present in many Hox genes and in hundreds of other chromosomal sites (Ferres-Marco, 2006).

Both polymerase chain reaction with reverse transcription (RT-PCR) and in situ hybridization experiments confirmed that transcription of lola and psq was influenced by eyeful in response to Gal4 activation (Ferres-Marco, 2006).

To determine whether lola and/or psq was responsible for the tumour phenotype, 11 enhancer promoter (EP) P-elements inserted into the lola and psq region were tested. In contrast to the GS lines, the EP lines allows Gal4-dependent inducible expression of sequences flanking only one end of the P-element. It was found that none of the EP lines induced tumours; thus, it was reasoned that the deregulation of both genes might be required to produce the tumours (Ferres-Marco, 2006).

The complexity of lola and psq loci, which together produce 23 proteins, hampers identification of the transcripts responsible for the eyeful phenotype by gain-of-expression mutants (that is, by expressing individual or combinations of isoforms). Therefore, this issue was resolved by isolating point mutations that reverted the phenotype caused by deregulated expression of lola and psq. In this analysis, the chemical mutagen ethyl-methane sulphonate (EMS) was used to induce preferentially single nucleotide changes (Ferres-Marco, 2006).

The parental eyeful GS line was viable in trans with deficiencies that removed both lola and psq. In contrast, a set of 14 EMS-induced mutations on the eyeful chromosome failed to complement these deficiencies and were found to be alleles of psq or lola. The EMS-induced mutations that best recovered a normal eye size were sequenced. Each individual mutation had a single base change or a small deletion that considerably altered the predicted Psq or Lola proteins (Ferres-Marco, 2006).

All psq- mutations induced on the eyeful chromosome prevented eyeful from producing eye tumours and metastases. Three alleles affected the BTB domain (psqrev2, psqrev7 and psqrev9), and three other alleles contained either a premature stop codon that would produce truncated proteins lacking the Psq HTH repeats (psqrev4 and psqrev14) or a missense mutation that would change a conserved amino acid in the third Psq HTH repeat (psqrev12). All lola- mutations induced on the eyeful chromosome, including the presumptive null allele (lolarev6), reduced eye tumour size but still permitted sporadic secondary growth (Ferres-Marco, 2006).

These data show an unequal contribution of Psq and Lola in this process, whereby Psq is the most important factor in the tumorigenic phenotype. The BTB subfamily of transcriptional repressors includes the human oncogenes BCL6 and PLZF. In these oncogenes, the BTB domain is crucial for oncogenesis through the recruitment of PcG and HDAC complexes. It is therefore speculated that deregulated Psq and Lola could lead to tumorigenesis by epigenetic processes and that Drosophila counterparts of HDACs and PcG proteins might be involved in the progression of these tumours. Indeed, genetic evidence was found that both Lola and Psq function as epigenetic silencers in vivo (Ferres-Marco, 2006).

Attempts were made to determine the specific epigenetic mechanisms through which deregulation of Psq and Lola might induce tumorigenesis in conjunction with Delta overexpression. Methylation of histone on lysine is a central modification in both epigenetic gene control and in large-scale chromatin structural organization. For example, trimethylation of histone H3 on K4 (H3K4me3) is associated with the active transcription of genes and open chromatin structure. By contrast, histone hypoacetylation and H3K9 and H3K27 methylation are characteristic of heterochromatin state and gene silencing. To determine whether any changes in these epigenetic markers might coincide with the induction of tumorigenesis, eye discs were immunolabelled with antibodies against specific histone H3 modifications. Because dorsal eye disc cells are refractory to Delta, the dorsal region of the discs provided an internal control for these studies (Ferres-Marco, 2006).

With the exception of some scattered cells, a prominent loss or strong reduction of H3K4me3 was observed in the ventral region of the mutant discs. Notably, although the loss of Notch in clones does not affect this epigenetic tag, overexpression of Delta caused a significant reduction in staining for H3K4me3. The H3K4me3 depletion was already apparent in discs showing moderate hyperplasia and thus preceded neoplasm formation. Changes in other epigenetic tags (such H3K9me3 and H3K27me2) could not be reproducibly resolved; perhaps more sensitive methods or antibodies might facilitate detection of such changes (Ferres-Marco, 2006).

H3K4 methylation is thought to be permissive for maintaining and propagating activated chromatin and is thought to neutralize repressor tags by precluding binding of the HDAC complex and impairing SUV39H1-mediated H3K9 methylation. Thus, H3K4me3 depletion may contribute to tumour formation by permitting aberrant chromatin silencing. It was found that a 50% reduction in dosage of the HDAC gene Rpd3 or of Su(var)3-9 decreased the tumour phenotype dominantly. In contrast, reducing the activity of the H3K4 histone methyltransferase genes Trx (known as ALL1 or MLL in humans) or Ash1, which would be expected to deplete the H3K4me3 tag further, did not visibly enhance the tumours (Ferres-Marco, 2006).

E(z) when complexed with the Extra sex combs (Esc) protein becomes a histone methyltransferase. The E(z)-Esc complex and its mammalian counterpart Ezh2-Eed show specificity for H3K27 but may also target H3K9. The complex also contains the HDAC Rpd3, and this association with Rpd3 is conserved in mammals. H3K27 methylation facilitates binding of the chromodomain protein Pc (HPC in humans), which then creates a repressive chromatin state that is a stable silencer of genes (Ferres-Marco, 2006).

Although loss of E(z) does not cause proliferation defects within discs, halving the E(z) gene dosage dominantly suppressed tumorigenesis, indicating that histone methylation by the E(z)-Esc complex is also a prerequisite for the excessive proliferation of these tumours. Accordingly, Esc- or Pc- mutations also notably reduced the tumours (Ferres-Marco, 2006).

Together, these findings suggest that the development of these tumours involves, at least in part, changes in the structure of chromatin brought about by covalent modifications of histones. These changes probably switch the target genes from the active H3K4me3 state to a deacetylated H3K9 and H3K27 methylation silent state (Ferres-Marco, 2006).

From this above data, it is considered that the tumours might form as a result of aberrant gene silencing. If so, then the expression of genes involved in cell-cycle control is likely to be altered in the mutant cells. The transcription of 12 tumour-related genes in the mutant and wild-type discs was compared. Transcription of the gene Rbf, a fly homologue of the RB/Rb family of genes, was strongly downregulated in this assay (and even in ey-Gal4 > Dl flies). A second Rb gene, Rbf2, remained unchanged in the different genetic backgrounds, highlighting the specificity of Rbf silencing (Ferres-Marco, 2006).

Inactivation of RB1 in retinoblastoma, a form of eye cancer in children, can occur through DNA hypermethylation of the promoter. Unlike in mammals, however, there is little cytosine methylation of the genome in Drosophila during developmental stages, and its potential role during tumorigenesis is unknown. DNA methylation seems to depend on one DNA methyltransferase, Dnmt2, that preferentially methylates cytosine at CpT or CpA sites. The fly genome also encodes one methyl-CpG DNA-binding MBD2/3 protein. Because there are no known Dnmt2 loss-of-function mutations, the role of this gene in tumorigenesis could not be tested (Ferres-Marco, 2006).

Nevertheless, whether the CpG islands that were observed in the Rbf gene were potential targets for repression by DNA methylation was tested by two methods. (1) Methylation-sensitive restriction enzymes analysis was used; this showed that the regions around the promoter and transcription start site of the Rbf gene are susceptible to methylation. This approach showed aberrant DNA hypermethylation of Rbf in eyeful and Delta eye discs and mild hypermethylation in Delta discs; however, at best only very mild methylation was detected in discs from wild-type flies or from flies with the control psq gene (Ferres-Marco, 2006).

(2) Direct bisulphite sequencing of genomic DNA was carried out from mutant discs. This approach confirmed the notable increase in methylated DNA in eyeful and Delta discs when compared with wild-type discs (and a moderate increase in methylated DNA in the Delta discs). Hypermethylation of the Rbf promoter was not simply the result of de novo transcription of Dnmt2 (ey-Gal4 > Dnmt2), indicating that activation of the Notch pathway is a crucial step in this de novo hypermethylation of Rbf (Ferres-Marco, 2006).

This study has used Drosophila genetics to search for genes that collaborate with the Notch pathway during tumorigenesis in vivo. Psq and Lola were identified as decisive factors to foment tumour growth and invasion when coactivated with the Notch pathway. These proteins are presumptive transcription repressors that contain a BTB domain and sequence-specific DNA-binding motifs and behave as epigenetic silencers in vivo (Ferres-Marco, 2006).

In addition, crosstalk between the Notch pathway and different epigenetic regulators was identified. It is likely that alterations in this crosstalk provoke the aberrant epigenetic repression (and perhaps also derepression) of genes that contribute to cellular transformation. The Rbf gene was identified as one target for this epigenetic regulation and Rbf depletion was shown to directly contribute to the tumours (Ferres-Marco, 2006).

It is proposed that the sequence of events that leads to these tumours commences with hyperactivation of the Notch pathway, which initiates gene repression. Subsequently, or at the same time as Notch, Psq-Lola could bind to the silenced genes and enforce silencing by recruitment of HDAC or PcG repressors. Given the conservation of the Psq-like HTH domains in Psq and of BTB domains, it seems likely that other transcriptional repressors containing such domains strongly influence the tumour-inducing capacities of HDACs and PcG repressors in human cancers (Ferres-Marco, 2006).

Finally, the collaboration between PcG-mediated cellular memory and the Notch pathway may have implications in other processes controlled by Notch, including the second mitotic wave in the Drosophila eye, and the organization of eye and wing growth. In these processes, the memory mechanism could ensure that cells keep a record of the Notch signals received at an earlier stage or when the progenitor cells were closer to the Delta source. In this way, they might remain proliferative without having to receive continuous instructions from Notch. Likewise, such a situation could be conceived for tumorigenesis. The oncogenic signals could opportunistically take advantage of the memory mechanism to fix and to maintain their instructions of continuous proliferation in progenitor or stem cells, thereby fostering tumour growth and metastasis (Ferres-Marco, 2006).

Delta and Egfr expression are regulated by Importin-7/Moleskin in Drosophila wing development

Drosophila DIM-7 (encoded by the moleskin gene, msk) is the orthologue of vertebrate Importin-7. Both Importin-7 and Msk/DIM-7 function as nuclear import cofactors, and have been implicated in the control of multiple signal transduction pathways, including the direct nuclear import of the activated (phosphorylated) form of MAP kinase. Two genetic deficiency screens were performed to identify deficiencies that similarly modified Msk overexpression phenotypes in both eyes and wings. Eleven total deficiencies were identifed, one of which removes the Delta locus. This report shows that Delta loss-of-function alleles dominantly suppress Msk gain-of-function phenotypes in the developing wing. Msk overexpression increases both Delta protein expression and Delta transcription, though Msk expression alone is not sufficient to activate Delta protein function. It was also found that Msk overexpression increases Egfr protein levels, and that msk gene function is required for proper Egfr expression in both developing wings and eyes. These results indicate a novel function for Msk in Egfr expression. The implications of these data are discussed with respect to the integration of Egfr and Delta/Notch signaling, specifically through the control of MAP kinase subcellular localization (Vrailas-Mortimer, 2007).

This study has focused on the effects of mutation in Delta, which was identified in this screen. Loss-of-function mutations in Delta dominantly suppress Msk over-expression phenotypes in developing wings. Further, Delta transcription and Delta protein expression is increased in areas over-expressing Msk protein in developing wing discs. Interestingly, the increased Delta protein induced by Msk over-expression is not competent to activate Notch signaling in adjacent cells. Thus, some mechanism must either be inhibiting this induced Dl protein from signaling to adjacent cells, or the induced Dl protein itself is non-functional for signaling (Vrailas-Mortimer, 2007).

Delta must be endocytosed in signal-sending cells in order to activate Notch in signal-receiving cells. Clones of cells that express Dl but are also deficient for Epsin, an adapter protein required for clathrin-mediated endocytosis, similarly can not promote Notch signaling in adjacent cells. It has been proposed that the Delta protein must normally be endocytosed and mono-ubiquitinated in the signal-sending cells (Delta expressing cells), where it is then targeted to a special endocytic pathway where it acquires competency to activate Notch in signal receiving cells. Thus, over-expression of Msk may have some effect on the internalization and/or post-translational modification of Delta (mono-ubiquitination) to render it unable to signal to adjacent cells. Indeed, Msk protein expression in en::msk wing discs is in a pattern that is co-incident with disrupted Delta protein near the apical tips of cells in the wing disc. Msk expression has been observed in the apical tips of cells within the morphogenetic furrow in the developing eye disc (Vrailas, 2006), where this apical localization is proposed to functionally inactivate Msk nuclear translocation function. Thus, in en::msk wing discs, apical localization of Msk protein may disrupt important cellular functions at this localization in the cell, such as Dl internalization and/or compartmentalization (Vrailas-Mortimer, 2007).

It has been shown that levels of over-expressed exogenous Delta in clones of cells is several fold higher than normal peak levels of endogenous Dl protein expression, and this over-expression autonomously inhibits Notch activation within these clones. This study also observed autonomous inhibition of Notch activation in posterior compartment cells that over-express Dl (UAS:Dl) with en:GAL4 (en::Dl), as measured by decreased Ct protein expression. Thus, the increased levels of Dl protein observed in en::msk wing discs may also explain the decrease in Ct protein expression in these wing pouches. However, increased Ct protein expression was also observed in posterior/dorsal cells when both exogenous Msk and exogenous Dl are over-expressed simultaneously. What can explain these apparently paradoxical results (Vrailas-Mortimer, 2007)?

It is known that the ectopic Dl protein induced by Msk over-expression in wings is unable to signal to adjacent cells. However, if this ectopic Dl expression is sufficient to autonomously inhibit Notch signaling in these cells (as observed by a decrease in Ct protein expression), it may function in a dominant-negative fashion in some cells but not in others. Thus, when both Msk and Dl are over-expressed, two things happen: (1) functional Dl protein is expressed that is competent to signal to adjacent cells (UAS:Dl); (2) non-functional Dl protein is expressed that is not competent to signal to adjacent cells, but is capable of autonomously inhibiting competent Dl protein (UAS:msk). There would then exist a situation within these cells where these two forms of Dl could compete for function. In those cells where competent Dl (UAS:Dl) wins, Ct expression is inhibited. In those cells where non-competent Dl (UAS:msk) wins, Ct expression can then be induced by competent Dl (UAS:Dl) expression in adjacent cells. This could account for the spotty appearance of Ct protein expression observed in these discs (Vrailas-Mortimer, 2007).

This study has shown that Egfr levels are decreased in msk clones in both larval wings and eyes, while Egfr levels are increased when Msk is over-express in larval tissues. These data suggest the possibility of a regulatory feedback mechanism on Egfr protein expression in this tissue. Thus, in cells where MAPK can move into the nucleus, the initial activation of the Egfr/Ras/MAPK pathway leads to the nuclear translocation of MAPK in these cells, which subsequently results in further upregulation of Egfr levels in those cells. This increased Egfr expression then further promotes even greater MAPK nuclear translocation in those cells. In cells where pMAPK is held in the cytoplasm, Egfr levels are decreased, and this may act as a feedback signal for continued hold of pMAPK within the cytoplasm of these cells. Indeed, Egfr mRNA expression is reduced in developing pupal wings after hyper-activation of Egfr signaling by rhomboid (rho) overexpression (rho encodes a protease required to activate the positive ligand spitz). The pMAPK induced by rho overexpression in developing pupal wings is also predominantly cytoplasmic, and leads to extra wing vein formation. Thus, the regulation of Egf receptor levels may be a mechanism by which subsequent MAPK subcellular localization is controlled (Vrailas-Mortimer, 2007).

How could the subcellular localization of MAPK relate to Dl expression and function in developing Drosophila tissues? In clones of spitz (which encodes for an activating ligand for the Egfr pathway) Dl expression is lost in the developing eye. Similarly, clones of cells mutant for the Egfr receptor itself show a loss of Dl expression in the developing pupal eye, although these clones show normal Cut protein expression. In the developing larval and pupal wing discs, Dl mRNA expression is absent in wing tissue double mutant for both rhomboid and vein (which effectively eliminates both the Egfr activating ligands spitz and vein in this tissue). Thus, Egfr activation and signaling are clearly required for Dl expression in these developing Drosophila tissues (Vrailas-Mortimer, 2007).

Dl expression is not lost in msk clones, suggesting: (1) the nuclear translocation of pMAPK is not required for Dl expression, (2) there is a redundant pMAPK nuclear transporter capable of importing pMAPK in these cells, (3) there is sufficient pMAPK nuclear translocation even in the absence of Msk protein to allow Dl expression to occur. Indeed, msk null clones posterior to the morphogenetic furrow in the developing eye retain many important Egfr/Ras pathway functions (Vrailas, 2006). Yet, over-expression of Msk increases both Dl protein expression and Dl transcription, suggesting that the nuclear translocation of pMAPK is at least sufficient to increase Dl protein levels. However, the Dl induced by Msk over-expression is not competent to activate Notch signaling in adjacent cells, suggesting that the nuclear translocation of pMAPK alone is not sufficient to induce Notch signaling in adjacent cells. In wild type wing cells, where high levels of competent, active Dl protein expression occur, high levels of phosporylated, cytoplasmic MAPK, and low levels of Egfr protein expression are also observed. Similarly, where high levels of Notch expression are observed, observe high levels of Egfr protein expression are also observed. Gain-of-function mutations in Notch, or hyper-activation of the downstream Notch protein Enhancer of split (E(spl)) decrease rho expression, while loss-of-function mutations in Notch, or expression of a dominant-negative form of Notch increases rho expression and induces extra vein formation. pMAPK expression is also lost upon loss of rho expression. Thus, Notch signaling represses pMAPK expression. As the pMAPK expression induced by rho signaling is predominantly cytoplasmic, it is suggested that it may be the cytoplasmic hold of pMAPK that is normally required for Dl protein signaling competence to activate Notch in adjacent cells. When competent Dl protein was overexpressed in the posterior compartment of developing wings (en:Gal4, UAS:Dl), Notch activation was induce in adjacent anterior/dorsal cells, and also increased expression of pMAPK was induced in the posterior compartment. It has previously been shown that pMAPK expression is lost in the posterior compartment of en::msk developing wing discs, since this pMAPK is ectopically translocated to the nucleus (Marenda, 2006). If pMAPK expression is required to induce Dl signaling competency, the difference in pMAPK expression observed between these two genotypes (en::msk and en::Dl) may explain the differences in Ct expression observed within these different genotypes as well (Vrailas-Mortimer, 2007).

Understanding how diverse signaling pathways integrate to regulate important biological processes is central to an understanding of the mechanisms of development. Understand these basic mechanisms of regulation and how they function to coordinately control different cellular processes are beginning to be understood. This report suggests that the subcellular localization of one pathway component (MAPK) as mediated by the nuclear import cofactor Msk, is an important factor in Egfr signal regulation through the control of the expression of the Egfr protein itself. It is further suggested that MAPK subcellular localization also plays an important role in the cross-talk between Egfr and Notch signaling pathways (Vrailas-Mortimer, 2007).

Delta function and hingut development

The Drosophila hindgut develops three morphologically distinct regions along its anteroposterior axis: small intestine, large intestine and rectum.
Single-cell rings of 'boundary cells' delimit the large intestine from the small intestine at the anterior, and the rectum at the posterior. The large intestine
also forms distinct dorsal and ventral regions; these are separated by two single-cell rows of boundary cells. Boundary cells are distinguished by their
elongated morphology, high level of both apical and cytoplasmic Crb protein, and gene expression program. During embryogenesis, the boundary cell
rows arise at the juxtaposition of a domain of Engrailed- plus Invected-expressing cells with a domain of Delta (Dl)-expressing cells. Analysis
of loss-of-function and ectopic expression phenotypes shows that the domain of Dl-expressing cells is defined by En/Inv repression. Further, Notch
pathway signaling, specifically the juxtaposition of Dl-expressing and Dl-non-expressing cells, is required to specify the rows of boundary cells. This
Notch-induced cell specification is distinguished by the fact that it does not appear to utilize the ligand Serrate and the modulator Fringe (Iwaki, 2002).

At its anterior, the hindgut joins the posterior midgut; at its posterior, it forms the anus. Along this AP axis, the hindgut of the mature embryo consists of three morphologically distinct domains: the wide, looping small intestine, the long and narrow large intestine, and the tapered rectum. Beginning at stage 13, these domains are demarcated at their junctions by rings of unusually high accumulation of the apical surface protein Crumbs (Crb). The ring at the small intestine/large intestine junction is designated the anterior boundary cell ring, and the ring at the large intestine/rectum junction is designated the posterior boundary cell ring (Iwaki, 2002).

Patterning of the hindgut in the DV axis is detected at stage 10 (germ band extension) when the hindgut develops an interiorly directed (dorsal) convexity. The side of the hindgut closest to the interior of the embryo is dorsal and expresses both En and Inv; that closest to the exterior is ventral and expresses dpp. By the completion of germ band retraction, the convexity at the anterior of the hindgut has shifted toward the left side of the embryo. Thus at the anterior of the hindgut, the initially dorsal, En- and Inv-expressing side comes to lie on the outer (left-facing) curve, while the initially ventral, Dpp-expressing side of the hindgut comes to lie on the inner (right-facing) curve; the DV relationship is retained at the posterior connection to the rectum. These initially DV patterned domains of the large intestine persist to the end of embryogenesis and into the larval stages; they are referred to as large intestine dorsal (li-d) and large intestine ventral (li-v). At each of the two boundaries between li-d and li-v, there is a single row of cells with high levels of Crb expression running the length of the large intestine, from the anterior boundary cell ring to the posterior boundary cell ring. These are designated the 'boundary cell rows'. In addition to their high level of Crb expression, the boundary cell rows and rings express the nuclear protein Dead ringer (Dri). Double antibody staining reveals that boundary cell rows at the border of the En/Inv-expressing li-d domain and the Dpp-expressing li-v domain express Dri in their nuclei and have strong Crb expression at their apical surfaces (Iwaki, 2002).

In addition to expressing Dpp, the li-v domain expresses the Notch ligand Delta (Dl); Dl is also expressed in the anterior of both the rectum and the small intestine. Fringe (Fng), a modulator of Notch signaling, is expressed opposite Dl in the Drosophila wing and eye; in the hindgut, Fng is expressed in li-d and the boundary cell rows, opposite the domain of Dl expression in li-d (Iwaki, 2002).

Interestingly, the Dri- and Crb-expressing boundary cells delimit both AP and DV boundaries in the hindgut. The rings form borders at the anterior and posterior ends of the large intestine, while the rows form borders between the dorsal (li-d) and ventral (li-v) regions of the large intestine. This study focusses primarily on the establishment and characteristics of the boundary cell rows (Iwaki, 2002).

Staining with both anti-Crb and anti-ßHEAVY Spectrin shows that the boundary cell rows are significantly more elongated along the AP axis than other hindgut epithelial cells. Staining of bynapro/+ embryos (containing a P-element insert in byn) with anti-ß-Gal antibody reveals that the nuclei of the cells of the boundary rows (identified by strong staining with anti-Crb) are also elongated in the AP axis (Iwaki, 2002).

The dramatically higher level of Crb expression in the boundary cells (both rings and rows) suggests that their apical surface may differ from that of other hindgut epithelial cells, and/or that, in the boundary cells, Crb may be present in cellular compartments in addition to the apical surface. Both of these expectations are borne out by a higher magnification examination of the boundary cells. In cross-sections of the large intestine viewed by electron microscopy, short microvilli on the apical surfaces of two cells on opposite sides of the hindgut lumen were observed; these cells most likely correspond to the boundary cell rows. The microvilli of the presumed boundary cell rows appear more organized and parallel than the irregular protrusions on the surfaces of the other cells of the hindgut epithelium. Because of their apical microvilli, the presumed boundary cell rows have a larger apical membrane surface and are expected to be labeled more strongly with anti-Crb. Consistent with this, cross-sections of anti-Crb-stained embryos viewed by light microscopy reveal two cells on opposite sides of the large intestine lumen with a higher level of Crb on their apical surfaces. In addition to their stronger apical labeling with anti-Crb, these presumed boundary cell rows also display an accumulation of Crb in their cytoplasm; this is strongest apical to the nucleus. The cytoplasmic accumulation of Crb suggests that Crb is produced at a higher level, or is more stable, in the boundary cells (Iwaki, 2002).

In conclusion, differences in gene expression demonstrate that the boundary cells are a separately patterned (fated) group of cells in the large intestine. The unique fate of the boundary cells is manifested both molecularly, in their expression of Dri and high cytoplasmic accumulation of Crb, and morphologically, in their marked AP elongation and development of apical microvilli (Iwaki, 2002).

The boundary cell rows form at the junction of the li-d and li-v domains, which express different genes. To investigate whether the spatially restricted gene expression observed in these domains is essential for establishment of boundary cell rows, embryos homozygous for loss-of-function alleles of en, inv, dpp, dri, Dl, Ser, Notch, or fng were examined. The presence or absence of boundary cells was assessed by anti-Crb staining, since this delineates their characteristic morphology, and also detects one of their unique differentiated features (i.e. the cytoplasmic accumulation of Crb) (Iwaki, 2002).

In embryos homozygous for a strongly hypomorphic dri allele (dri null mutants lack a discernable hindgut), the hindgut is of roughly normal diameter but only about one-third its normal length. Even in these severely reduced dri hindguts, however, boundary cells can still be observed; this phenotype is similar to that described for embryos lacking both maternal and zygotic dri function. Since reduced hindgut size is observed in embryos that lack zygotic, but retain maternal dri function, it is concluded that zygotic expression of dri (most likely the uniform expression at the blastoderm stage) is required to establish or to maintain the normal-size hindgut primordium. Neither blastoderm expression of dri, nor its later expression in the boundary cells, however, appears to be required to establish the boundary cells (Iwaki, 2002).

In dpp embryos, the large intestine is shorter; this is believed to be due to a requirement for dpp in DNA endoreplication in the large intestine. Although the hindgut is variable and severely deformed in dpp mutant embryos (only rudimentary hindguts are detected in the strongest dpp alleles), boundary cell rows were detectable in the hindguts of embryos carrying several different strongly hypomorphic dpp alleles. Thus even though it is required for normal hindgut development, dpp activity does not appear to be required to establish the boundary cell rows (Iwaki, 2002).

In embryos lacking only en, the boundary cell rows and rings form normally. Similarly, many embryos lacking only inv form boundary cell rows and rings. In a significant number of inv embryos, however, gaps were observed in the posterior of the boundary cell rows. This is the only embryonic phenotype known for inv. When both en and inv are removed [in Df(enE) embryos], the phenotype is much more dramatic: boundary cell rows and rings are completely absent. Consistent with previous studies demonstrating a functional redundancy of en and inv, it is concluded that en and inv are required largely redundantly to establish the boundary cells. However, while inv can substitute completely for en, there is a requirement for inv that cannot be completely substituted by en. This is likely not due to a difference in protein structure, but rather to the fact that, in the hindgut, inv is expressed earlier and at a higher level than en. As their functions are so closely intertwined, the activities of en and inv, and the highly related proteins that they encode, are referred to as single entities: en/inv and En/Inv (Iwaki, 2002).

Embryos lacking Dl function are extremely deformed and do not always have a recognizable hindgut, indicating that function of Dl early in embryogenesis is required to establish and/or maintain the hindgut. Since Dl encodes a ligand for Notch, embryos lacking the zygotic contribution of Notch were examined. Strikingly, Notch mutant hindguts completely lack both boundary cell rows and rings, revealing that Notch signaling is required to establish the boundary cells. The data demonstrate that formation of the boundary cell rows at the border of Dl expression requires the Notch receptor; however, Fng does not appear to be required for this process (Iwaki, 2002).

To further investigate the required role of Dl in establishing the boundary cells, a dominant-negative form of Dl was expressed throughout the hindgut. bynGal4:UAS-Dl.DN embryos show a complete absence of boundary cell rows and rings; this phenotype closely resembles that seen in Notch loss-of-function embryos. Expression of a dominant negative Notch receptor throughout the hindgut results in a similar absence of boundary cell rows and rings. Furthermore, bynGal4 driven expression of UAS-Hairless, which acts to suppress activity of Su(H) also results in an absence of boundary cells. This last result indicates that the Notch signaling required to establish the boundary cells must act through Su(H). In summary, the above results demonstrate required roles in boundary cell specification of the following Notch pathway components: the ligand Dl, the receptor Notch, and the downstream transcription factor Su(H). It is therefore concluded that the Notch signaling pathway is required for boundary cell induction (Iwaki, 2002).

An intriguing observation, given the demonstrated role of the LIN-12/Notch signaling pathway in generation of left¯right asymmetry in the Caenorhabditis elegans intestine is that a large portion of 455.2Gal4:UAS¯Su(H)VP16 hindguts display a reversal of left¯right looping (Iwaki, 2002).

Ectopic expression experiments, taken together with the loss-of-function experiments, demonstrate that establishment of the boundary cell rows requires the juxtaposition of Dl-expressing and Dl-non-expressing cells and signaling via Notch and Su(H). In addition to Notch and spatially restricted Dl, establishment of the anterior ring requires localized activity of Dpp; the posterior ring requires En/Inv activity (which does not need to be localized) and the localized activity of Dl (Iwaki, 2002).

Since the experiments described in the preceding sections show that both spatially localized En/Inv and a boundary of Dl expression are required to establish the boundary cells, it was asked whether En/Inv might control the boundary of Dl expression. In Df(enE) embryos, Dl is not restricted to li-v, but rather is uniform in the hindgut circumference, indicating that en/inv is required to repress Dl. In the large intestine, uniform expression of En/Inv results in an absence of Dl expression. Expression of En/Inv in li-d is thus both necessary and sufficient to restrict Dl expression to li-d. While it represses Dl throughout the large intestine, ectopic En/Inv does not affect Dl expression in the rectum. Embryos with ectopic En/Inv not only express Dl at the anterior of the rectum, they also form the posterior boundary cell ring. Thus a boundary of Dl-expressing with Dl-non-expressing cells is required not only to establish the boundary cell rows but also likely to establish the posterior ring; the posterior ring also requires En/Inv activity, but this activity does not need to be localized (Iwaki, 2002).

Consistent with observations that En and Inv are repressors with the same targets, the data presented in this study demonstrate that Dl expression in the large intestine is restricted to the li-v domain by the repressive activity of En/Inv in li-d (Iwaki, 2002).

The data presented here support the following model. En/Inv is expressed in li-d and represses Dl in that domain; Dl expression is thereby restricted to the li-v domain. At the li-v/li-d transition, the Dl-expressing cells induce, by Notch signaling, a row of Dl-non-expressing cells to become a boundary cell row. Since En/Inv is not detected in differentiated boundary cells, Notch activation likely represses En/Inv expression. Notch activation also leads to Dri expression and an upregulation of Crb expression. While all of these transcriptional changes could be mediated by Su(H), they could also be further downstream (Iwaki, 2002).

In summary, three steps in the establishment of the Drosophila hindgut boundary cell rows are similar to steps characterized in other Notch dependent boundary-forming systems. (1) A homeodomain transcription factor (En/Inv in the case of the boundary cells) is expressed on one side of the forming boundary; (2) this transcription factor defines two domains, one which expresses Dl and one which does not; (3) Notch activation in the Dl-non-expressing cells that confront Dl-expressing cells leads to a unique cell fate (Iwaki, 2002).

Given the essential role of spatially restricted En/Inv expression in establishing the boundary cells, it is of interest to consider how En/Inv expression is restricted to the li-d domain. The activation of en expression in the large intestine at stage 10 requires the T-domain transcription factor brachyenteron (byn), which is expressed uniformly in the hindgut. Since dissection of the en regulatory region has identified fragments that drive reporter expression in all hindgut cells, en expression is likely restricted to li-d by a repressor that remains to be identified (Iwaki, 2002).

Boundary cells could be imagined to provide adhesive differences important for cell rearrangement; alternatively, their AP elongation might provide a mechanical force to drive hindgut elongation. In spite of these tempting scenarios, however, the normal appearance (overall size, diameter, and length) of Notch and Df(enE) hindguts, which completely lack both boundary cell rows and rings, demonstrates conclusively that the boundary cell rows and rings are not required to establish normal hindgut morphology (Iwaki, 2002).

Rather than playing a required role in hindgut morphogenesis, the boundary cells most likely contribute to the ion and water absorption function of the larval hindgut. In the adult insect, this function is carried out by cells in the rectum that are distinguished by their extensive, mitochondria-rich apical membrane leaflets. In the Drosophila larval hindgut, this characteristic ultrastructure is found not in the rectum, but rather in the cells of li-d, leading to the conclusion that water and ion absorption in the larva occurs in the large intestine. Associated with the absorptive cells of the Dipteran rectum is a distinct cell type referred to as 'junctional cells'; these form a collar surrounding the absorptive cells, have extensive intercellular junctional complexes, and are thought to play an isolating and supportive role. The Drosophila boundary cell rings and rows similarly constitute a collar surrounding the absorptive li-d cells of the larval hindgut and, based on their intensive Crb staining, have unusual membrane characteristics. It is therefore proposed that, like the junctional cells in the adult insect rectum, the boundary cells serve to isolate and support a domain of ion and water absorbing cells in the Drosophila larval hindgut (Iwaki, 2002).

Delta and axon guidance

The central problem in axon guidance is to understand how guidance signals interact to determine where an axon will grow. A specific axon guidance decision in Drosophila embryos has been investigated, the sharp inward turn taken by the ISNb motor nerve to approach its muscle targets. This turn requires Notch and its ligand Delta. Delta is expressed on cells adjacent to the ISNb turning point, and it is known from previous work that Notch is present on axonal growth cones, suggesting that Delta and Notch might provide a guidance signal to ISNb. To induce the turning of ISNb axons, Notch interacts genetically with multiple components of a signal transduction pathway that includes the Abl tyrosine kinase and its affiliated accessory proteins. In contrast, genetic interaction experiments fail to provide evidence for a major role of the 'canonical' Notch/Su(H) signaling pathway in this process. It is suggested that the Notch/Abl interaction promotes the turning of ISNb axons by attenuating the Abl-dependent adhesion of ISNb axons to their substratum, thus releasing the axons to respond to attraction from target muscles (Crowner, 2003).

The receptor Notch is present on axons and growth cones and is required for extension of some early-growing 'pioneer' axons in the fly embryo. More recently, later functions of Notch in axon patterning have been investigated, using a temperature-sensitive Notch allele (Notchts1) to remove Notch activity well after most embryonic neuronal identities have been specified. In temperature-shifted mutant embryos, it has been found that ISNb axons reach their targets via an aberrant bypass trajectory, in which ISNb axons remain associated with the ISN. All Notchts embryos display the bypass phenotype, with 31% of hemisegments affected. Raising the temperature 1 hr earlier in development increases the expressivity of the ISNb bypass phenotype to 73% of hemisegments. Wild-type embryos subjected to the same temperature protocol, or Nts embryos maintained at 25°, displayed few if any defects in ISNb defasciculation. Despite the aberrant pathfinding in Notchts embryos, formation of neuromuscular synapses to ventral longitudinal muscles occur as efficiently in temperature-shifted mutant embryos as in similarly treated wild-types (Crowner, 2003).

The site of Notch activation was localized by examining the Notch ligand, Delta. Temperature shifts of a temperature-sensitive combination of Delta alleles (Dl6B37/Dlvia1) produced an ISNb bypass phenotype indistinguishable from that induced by Notchts. The Deltats mutant combination is not as 'tight' as Notchts1, so the reduced expressivity of the Delta phenotype relative to that of Notch is not surprising. Antibody staining revealed that at the time when ISNb is pioneered, Delta is expressed on cells very near to the first choice point, most prominently on the ganglionic branch of the trachea. This tracheal branch develops prior to ISNb outgrowth, the ISN grows in close association with the trachea, and ISNb axons separate from the ISN at that point where they first contact the trachea. The highest tracheal acccumulation of Delta protein is on the apical surface of the cells, in the tracheal lumen; however, Delta protein is also found on the basal surface of tracheal cells, available for interaction with ISNb axons. Adding back wild-type Delta to the trachea of temperature-shifted Deltats embryos (with btl-GAL4) rescues the Delta ISNb bypass phenotype. btl-GAL4 is expressed in midline glial cells in addition to tracheal cells; however, midline expression of Delta does not rescue ISNb trajectory in Deltats. Staining with an anti-tracheal antibody demonstrates that the ganglionic tracheal branch develops normally in temperature-shifted Nts embryos (Crowner, 2003).

While tracheal expression of Delta is sufficient to restore ISNb defasciculation, ISNb still defasciculates properly in btl mutant embryos that lack trachea. Delta protein, however, is also detectable on nontracheal cells that abut the first choice point, and it is postulated that the Delta on these other cells might act redundantly with that on the trachea to provide a defasciculation signal for ISNb axons. The positions of these cells are consistent with some of them being peripheral glia, and indeed some of these cells label with Repo, a marker for glial cell nuclei. Embryos lacking glia show a low frequency of ISNb bypasses, and this frequency was substantially enhanced in embryos that simultaneously lack the trachea, consistent with the notion that both glia and trachea contribute to the defasciculation of ISNb at the first choice point (Crowner, 2003).

Notch signaling mediates multiple developmental decisions in Drosophila. This study examined the role of Notch signaling in Drosophila larval optic lobe development. Loss of function in Notch or its ligand Delta leads to loss of the lamina and a smaller medulla. The neuroepithelial cells in the optic lobe in Notch or Delta mutant brains do not expand but instead differentiate prematurely into medulla neuroblasts, which lead to premature neurogenesis in the medulla. Clonal analyses of loss-of-function alleles for the pathway components, including N, Dl, Su(H), and E(spl)-C, indicate that the Delta/Notch/Su(H) pathway is required for both maintaining the neuroepithelial stem cells and inhibiting medulla neuroblast formation while E(spl)-C is only required for some aspects of the inhibition of medulla neuroblast formation. Conversely, Notch pathway overactivation promotes neuroepithelial cell expansion while suppressing medulla neuroblast formation and neurogenesis; numb loss of function mimics Notch overactivation, suggesting that Numb may inhibit Notch signaling activity in the optic lobe neuroepithelial cells. Thus, these results show that Notch signaling plays a dual role in optic lobe development, by maintaining the neuroepithelial stem cells and promoting their expansion while inhibiting their differentiation into medulla neuroblasts. These roles of Notch signaling are strikingly similar to those of the JAK/STAT pathway in optic lobe development, raising the possibility that these pathways may collaborate to control neuroepithelial stem cell maintenance and expansion, and their differentiation into the progenitor cells (Wang, 2011).

This study find that Notch signaling plays an essential role in the maintenance and expansion of neuroepithelial cells in the optic lobe; it also inhibits medulla neuroblast formation. Clonal analyses of several pathway components indicate that this dual function bifurcates downstream of Su(H) with E(spl)-C only partly involved in the inhibition of medulla neuroblast formation but not the maintenance and expansion of neuroepithelial stem cells (Wang, 2011).

In the optic lobe, Notch signaling plays a role analogous to lateral inhibition during embryonic CNS development. However, the selection of neuroblasts in the OPC neuroepithelium is an all-or-none process rather than selecting individual neuroblasts from the neuroepithelium. Medulla neuroblasts are generated in a wave progressing in a medial to lateral direction in the OPC neuroepithelium with all cells at a particular position along the medial-lateral axis differentiating into neuroblasts. Interestingly, this wave of medulla neuroblast formation coincides with the down-regulation of both Delta and Notch expression in the medial cells in the OPC, which might reduce Notch signaling activity, thereby allowing medulla neuroblasts to form. What factors drive the recession of both Delta and Notch expression in the OPC neuroepithelium along the medial-lateral axis is not known. When Notch signaling is inactivated, neuroepithelial cells in the OPC change cell morphology and differentiate into medulla neuroblasts prematurely. The results indicate that Notch signaling actively controls neuroepithelial integrity, possibly by regulating the adherens junction (AJ), since in Notch pathway mutant mosaic clones in the OPC, the apical determinants PatJ, Crumbs and aPKC are cell autonomously reduced or lost and the mutant cells change to rounded or irregular morphology. Further experiments will be needed to determine how Notch signaling activity affects the maintenance of neuroepithelial integrity, particularly the stability of the adherens junction (Wang, 2011).

Is neuroblast formation also actively inhibited by Notch signaling or simply a default state of neurogenic epithelial cells? In the latter model, Notch signaling may only maintain neuroepithelial integrity and promote their expansion while medulla neuroblasts form when the neuroepithelial integrity is disrupted. The argument against this model is that changes in neuroepithelial integrity are not always accompanied with cell fate changes. In N, Dl or Su(H) mosaic clones located in the OPC neuroepithelium, it was found that in about 25% of the clones, the mutant cells changed morphology or lost apical marker expression but did not become neuroblasts (Dpn-negative), whereas in E(spl)-C mosaic clones, Dpn+ cells were prematurely induced, which indicate that the cells begin to differentiate into neuroblasts, but these cells still retained columnar epithelial cell morphology and apical marker expression. This suggests that the suppression of neuroblast formation by Notch signaling activity is separable from the maintenance of neuroepithelial integrity and that medulla neuroblast formation is actively suppressed by Notch signaling. A possible scenario is that activation of the Notch pathway turns on the E(spl)-C genes, which in turn suppress proneural gene expression in the optic lobe neuroepithelia. Indeed, at least one member in the E(spl)-C genes, E(spl)m8, appears to be activated in the neuroepithelial cells by the Notch pathway, as the E(spl)m8-lacZ reporter is expressed in a pattern similar to Delta and Notch expression in the OPC and IPC. E(spl)m8 protein and possibly additional members of the E(spl)-C may suppress the expression of proneural genes in the optic lobe. The proneural genes of the achaete-scute complex (as-c) comprise four members, achaete, scute, L'sc, and asense. achaete is not expressed in the optic lobe, but scute is expressed in both the neuroepithelial cells and neuroblasts in the OPC implying that scute expression in the neuroepithelial cells is not suppressed by Notch signaling activity. By contrast, asense is only expressed in the neuroblast and GMCs and L'sc is transiently detected in an advancing stripe of neuroepithelial cells of 1-2 cells wide that are just ahead of newly formed medulla neuroblasts. Thus, E(spl)-C proteins may suppress L'sc and/or ase expression, the release of this suppression may allow the neuroepithelial cells to begin to differentiate into medulla neuroblasts. It should be noted, however, that the removal of the E(spl)-C activity does not seem to be sufficient to allow full differentiation of neuroepithelial cells into medulla neuroblasts, suggesting that additional factors downstream of Notch signaling may be involved in the suppression of medulla neuroblast formation (Wang, 2011).

The phenotypes of Notch pathway mutants are reminiscent of those of JAK/STAT mutants. For example, inactivation of either pathway led to early depletion of the OPC neuroepithelium; either pathway inhibits neuroblast formation, and ectopic activation of either pathway promotes the growth of the OPC neuroepithelium. The remarkable phenotypic similarities in Notch and JAK signaling mutant brains suggest that these pathways may act in a linear relationship such that activation of one pathway is relayed to the second, perhaps by inducing the expression of a ligand. Alternatively, these pathways may act in parallel and converge onto some key downstream effectors or target genes. Further experiments will be needed to test whether Notch interacts with JAK/STAT and if it does, to find out where the interaction occurs during the development of the optic lobe (Wang, 2011).

The roles of Notch signaling in mammalian brain development have been studied intensely. Many Notch pathway components have been examined in knockout mice, which showed defects in brain development. Mice deficient for Notch1 or Cbf all display precocious neurogenesis during early stages of nervous system development. This has led to the view that the role of Notch signaling in the mouse brain is to maintain the progenitor state and inhibit neurogenesis. However, it is not clear from these studies whether the premature neurogenesis in Notch signaling mutant mice was caused by premature differentiation of neuroepithelial stem cells into neurons or by premature differentiation of neuroepithelial stem cells into progenitor cells, which then generated neurons. In fact, it has been proposed that Notch activation can promote the differentiation of neuroepithelial stem cells into radial glial cells, the progenitor cells that generate the majority of neurons in the cerebral cortex. This is based on the observation that ectopic Notch activation using activated forms of Notch1 and Notch3 (NICD) caused an increase in radial glial cells as compared to control. The radial glial cells resemble medulla neuroblasts in the Drosophila optic lobe in that they are both derived from neuroepithelial stem cells and undergo asymmetric division to self-renew and generate neurons, although morphologically radial glial cells are still polarized while medulla neuroblasts have lost epithelial characters and are rounded in shape. Based on the current results, it is suggested that Notch signaling maintains the pool of neuroepithelial stem cells and promotes their expansion in both Drosophila and mammals and that the precocious neurogenesis in Notch signaling mutant brains arise due to premature differentiation of the neuroepithelial stem cells into the progenitor cells (Wang, 2011).

However, ectopic Notch activation may indeed promote progenitor cell proliferation in the brain. Ectopic neuroblasts were observed in the medulla cortex when NACT was ectopically expressed by the neuroblast/GMC driver insc-Gal4, by ubiquitous expression using hs-Gal4, or when numb15 mosaic clones were induced at later larval stages when neuroblasts normally begin to form. Since the results have shown that the Notch pathway is not essential for medulla neuroblast formation or self-renewal, the ectopic neuroblasts are a novel phenotype solely induced by ectopic Notch signaling activity. This is consistent with Notch activation promoting ectopic neuroblast formation in the central brain and VNC without being required for neuroblast self-renewal in these regions of the CNS; and Notch has been shown to be an oncogene in mammals. Since the sizes of the ectopic neuroblasts were in the range of GMC or neurons, they may resemble the transit-amplifying (TA) neuroblasts that are found in the dorsal-medial region of the central brain. The origin of these ectopic neuroblasts in the medulla cortex is not clear, but it is unlikely that they are derived from differentiated medulla neurons as ectopic expression of NACT using elav-Gal4, which is active in medulla neurons, did not result in ectopic neuroblasts and by the fact that ectopic neuroblasts can be induced in numb15 mosaic clones, which could only arise from mitotically active cells that include neuroepithelial cells, medulla neuroblasts, and ganglion mother cells (GMCs), but not neurons. The ectopic neuroblasts could be generated by a transformation of GMCs into a neuroblast identity as suggested for ectopic neuroblasts in brat mutant central brains. Ectopic Notch signaling activity may even directly promote the expansion of neuroblasts after they have differentiated from the neuroepithelial cells in the OPC. In either case, ectopic Notch signaling activity may block the normal path of neuronal differentiation and lock the cells in a proliferative state. This is indeed what was observed in numb15 mosaic clones in which numerous ectopic neuroblasts were induced in the medulla cortex without generating medulla neurons. Perhaps ectopic Notch signaling activity may also promote the proliferation of neural progenitors in vertebrates, such as the radial glial cells in the mouse brain (Wang, 2011).

The role of the transmembrane receptor Notch in the adult brain is poorly understood. This study provides evidence that bunched, a negative regulator of Notch, is involved in sleep homeostasis. Genetic evidence indicates that interfering with bunched activity in the mushroom bodies (MBs) abolishes sleep homeostasis. Combining bunched and Delta loss-of-function mutations rescues normal homeostasis, suggesting that Notch signaling may be involved in regulating sensitivity to sleep loss. Preventing the downregulation of Delta by overexpressing a wild-type transgene in MBs reduces sleep homeostasis and, importantly, prevents learning impairments induced by sleep deprivation. Similar resistance to sleep loss is observed with Notchspl-1 gain-of-function mutants. Immunohistochemistry reveals that the Notch receptor is expressed in glia, whereas Delta is localized in neurons. Importantly, the expression in glia of the intracellular domain of Notch, a dominant activated form of the receptor, is sufficient to prevent learning deficits after sleep deprivation. Together, these results identify a novel neuron-glia signaling pathway dependent on Notch and regulated by bunched. These data highlight the emerging role of neuron-glia interactions in regulating both sleep and learning impairments associated with sleep loss (Seugnet, 2011).

The evidence presented suggests that Notch signaling
controls factors that reduce the negative consequences of
waking as measured by an attenuated sleep rebound and
intact learning following 12 hr of sleep deprivation. Although
it is tempting to speculate that the intact learning seen
following sleep loss is simply due to the flies not being sleepy,
previous studies have shown that sleepiness does not
result in performance impairments in aversive phototaxic suppression (APS; Seugnet, 2008). Thus, Notch activation may preserve learning by preventing neuronal
overstimulation during extended waking. Reducing neuronal
stimulation may also prevent the buildup of sleep debt and
thus explain the lack of sleep rebound. Canonical Notch
signaling leads to Su(H)-dependent changes in transcription,
but several other downstream pathways have been identified; thus, further work is required to determine which pathway downstream of the receptor is effectively involved in this context. The results suggest that Notch is mediating
a neuron-glia signaling mechanism. These data provide additional
support to recent work showing an involvement of glia
in sleep homeostasis and cognitive impairments. In mammals, adenosine released by glia appears to play a critical role (Halassa, 2009). Given that mutants for the only known Drosophila adenosine receptor have normal sleep homeostasis (Wu, 2009), other factors are likely to be involved. It is interesting to note in this
context that expression of the cell adhesion molecule Klingon,
required for long-term memory and controlled by Notch in the
adult brain, has been reported to be expressed in the glia.
It should be noted that Notch localization and activation in glia
may seem at odds with reports showing a requirement for
Notch as well as the downstream effector Su(H) in MB neurons
for memory consolidation. The data do not exclude
a low level of Notch expression in neurons. In fact, it would
not be surprising if Notch is expressed in both cell types and
mediates two-way signaling between adjacent cells, given
that it occurs commonly during developmental processes (Seugnet, 2011).